Systems and methods for ordering, performing, and reporting genetic screening

ABSTRACT

The present invention provides computer-implemented systems and methods for ordering, performing, and reporting transgenic and targeted mutagenesis screening of genomic DNA. The methods are performed by a remote user&#39;s computer, a web site computer and a laboratory computer. The remote user, in communication with another computer, requests a screening of biological samples that are disposed in a multi-well plate. The remote user designates on his computer a variety of parameters necessary to perform the testing and identifies the location of samples in the multi-well plate. A computer at the laboratory receives this information and uses it to perform testing, and to prepare a sample test report that is returned to the remote user. A computer intermediate to the user&#39;s computer and the laboratory computer, such as a web site computer, may be used. The laboratory computer is configured to extract information from the remote user&#39;s electronic communication and transmit that information electronically to a supplier to order reagents necessary for the testing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 120 as a CONTINUATIONAPPLICATION of a co-pending application entitled “System, Method andApparatus for Transgenic and Targeted Mutagenesis Screening” which wasfiled on Sep. 4, 2001, and was assigned U.S. application Ser. No.09/945,952 (the “'952 application”), the entire disclosure of to whichis incorporated herein by reference for all that it teaches. Thisapplication and the '952 application also claim priority under 35 U.S.C.§119(e), based on U.S. Provisional Application Ser. No. 60/230,371,filed Sep. 6, 2000, the entire disclosure of which is incorporatedherein by reference for all that it teaches.

FIELD OF THE INVENTION

This invention relates to a system for transgenic and targetedmutagenesis screening. Additionally, this invention relates to variousmethods to detect or screen for designated genetic sequences or portionthereof derived from a tissue sample. More specifically, this inventionrelates to a high volume apparatus for transgenic and targetedmutagenesis screening.

BACKGROUND OF THE INVENTION

Genomic modification resulting from mutations in the DNA of an organismcan be transferred to the progeny if such mutations are present in thegametes of the organism, referred to as germ-line mutations. Thesemutations may arise from genetic manipulation of the DNA usingrecombinant DNA technology or may be introduced by challenging the DNAby chemical or physical means. DNA introduced via recombinant DNAtechnology can be derived from many sources, including but not limitedto DNA from viruses, mycoplasm, bacteria, fungi, yeast, and chordatesincluding mammals such as humans.

Recombinant DNA technology allows for the introduction, deletion orreplacement of DNA of an organism. Random introduction of DNA into acell can be achieved by technologies such as transfection (includingelectroporation, lipofection), injection (pronuclear injection, nucleartransplantation) or transduction (viral infection). Random mutations(point mutations, deletions, amplifications) can be generated bytreatment of cells with chemical mutagens or submitting them to physicalinsult such as X-irradiation or linear energy transfer irradiation(LET). Targeted addition, deletion or replacement of DNA in an organism(either inducible or non-inducible) is achieved via homologousrecombination. Inducible systems employ sequence-specific recombinasessuch as Cre-LoxP (U.S. Pat. Nos. 5,654,182 and 5,677,177) and FLP/FRT(U.S. Pat. No. 5,527,695) (hereby incorporated by reference).

Transgenic organisms are organisms that carry DNA sequences (be it genesor gene segments) derived from another species, stably integrated intotheir genome. Transgenic mammals are generally created by microinjectionof DNA into the pronucleus of fertilized eggs, a technique in which thenumber of DNA copies or the integration site of the DNA into the hostgenome is uncontrollable. A transgenic line refers to an organism thattransmits the foreign DNA sequences to its offspring.

Targeted mutations, site directed mutagenesis or gene targeting isdescribed as methods that employ homologous recombination of DNA toalter a specific DNA sequence within the host genome. This can result ininactivation of a gene (knock-out mutation), or genetic alteration ofthe gene (knock-in mutation). In mammals this can be achieved bytransfection of a cloned, mutated gene segment (targeting construct)into embryonic stem cells (ES cells), which, via homologousrecombination, replaces the endogenous gene segment in the ES cell.Animals derived from these ES cells will carry the targeted mutation intheir genome. Further refinement of this technique involves induciblegene alteration, in which the endogenous gene has been targeted with aDNA segment that contains recognition sequences (LoxP or FRT sequences)for site-specific recombinases (Cre, FLP). Expression of the recombinasein the targeted ES cell or the ES cell-derived animal will result indeletion of the DNA segment flanked by the recognition sites. Dependingon the configuration of targeting construct, this can result ininactivation, activation or alteration of the targeted gene. Theadvantage of an inducible system in animals is that the gene alterationcan be induced at any point in time or in any tissue, depending on theability to specifically activate the recombinase. This can be achievedby placing the recombinase under the control of inducible promoters(chemically or hormone-inducible promoters).

Transgenic and targeted mutagenesis screening is used to determine if agenome possesses specific genetic sequences that exist endogenously orhave been modified, mutated or genetically engineered. Genomic DNA isscreened for these modifications or mutations. Genomic DNA ischallenging to sufficiently immobilize on the substrate because of itssize. The genomic DNA includes both coding and non-coding regions.Therefore, the genomic DNA contains exons and introns, promoter and generegulation regions, telomeres, origins or replication and non-functionalintergenic DNA. The genomic DNA is a double stranded molecule which ismethylated. Immobilizing cDNA and PCR-amplicons differs in that themolecules are much smaller. Additionally, biochemical modificationevents, such as methylation, do not occur with the smaller molecules.Shena, M (2000) DNA Microarrays: A Practical Approach. Oxford UniversityPress, New York, N.Y. (hereby incorporated by reference).

Transgenic screening is currently done manually. The present manualsystem is time-consuming and can provide variable results depending onthe laboratory and even depending on skill of laboratory workers.Presently, a researcher using Southern blot technology may requiregreater than a week to screen a tissue sample for a transgene or atargeted mutation.

In an alternative technology, up to thirty PCR (polymerase chainreaction) can be conducted in an Eppendorf microtube® (BrinkmannInstruments, Westbury, N.Y.) and separated on a gel. This process inmost laboratories requires 3 to 7 days. A need exists in the industry toprovide a system and method for more accurate, faster and high volumetransgenic and targeted mutagenesis screening.

SUMMARY OF THE INVENTION

The present invention provides a unique solution to the above-describedproblems by providing a method and system for automated transgenic andtargeted mutagenesis testing.

The object of this invention is to provide higher volume screening oftransgenic and targeted mutagenesis samples for a designated geneticsequence than by prior art methods. It is another object of thisinvention to provide screening results to a researcher more quickly thanby prior art method to screen transgenic and targeted mutagenic samples.These objects are achieved by several features of this invention. Thesefeatures include depositing prokaryotic or eukaryotic genomic DNA on asubstrate and detecting the genomic DNA with a microarray imager tofacilitate high volume screening. Additionally, an order process thatprovides a remote user's selection parameters to conduct screening of asample and provides the associated reagents, in a coordinated wayfacilitates high volume screening of transgenic and targeted mutagenesissamples for a designated genetic sequence. In addition to this feature,screening of genomic DNA from cellular lysate using magnetic particlesand lysing the tissue sample with a lysis buffer that is formulated towork while the to samples are in transit to the screening laboratoryfrom a remote user have been found to facilitate high volume screening.It should be noted that the techniques taught in the specification thatenable higher volume screening of genomic DNA for a designated geneticsequence can be more broadly applied to by one skilled in the art tovarious methods to detect genetic sequences in samples of genomic DNA.

According to another aspect of this invention, a method is provided todetect a designated genetic sequence in a sample of genomic DNA. Thismethod involves depositing the genomic DNA on a substrate; adding atleast one labeled probe specific for a portion of the designated geneticsequence; and detecting the signal from at least one labeled probespecific for a portion of the designated genetic sequence to detect thedesignated genetic sequence in the sample of genomic DNA.

According to another aspect of this invention, a method is provided todetect a designated genetic sequence in a sample of genomic DNA, bycomparing the sample with a designated control sample of genomic DNA.This method involves the steps of: depositing the genomic DNA from thesample at a first location on the substrate; depositing genomic DNA fromthe designated control sample at a second location on the substrate;adding at least one labeled probe specific for a portion of thedesignated genetic sequence to the first and second locations on thesubstrate; detecting the signal from the at least one labeled probespecific for a portion of the designated genetic sequence at the firstand second locations on the substrate, and comparing the signal from thefirst and second locations on the substrate to detect a designatedgenetic sequence in the sample of genomic DNA.

In another aspect of this invention, a method is provided to detect adesignated genetic sequence in a sample of tissue by comparing thesample to a designated control sample of tissue. This method comprisesthe steps of: treating the sample of tissue and the designated controlsample of tissue with a sufficient amount of a lysis buffer to obtaincellular debris including genomic DNA; separating the genomic DNA fromthe cellular debris for the sample of tissue and the designated controlsample of tissue; depositing the genomic DNA from the sample at a firstlocation on a substrate; depositing the genomic DNA from the designatedcontrol sample at a second location on the substrate; adding at leastone labeled probe specific for a portion of the designated geneticsequence to the first and second locations on the substrate; detectingthe signal from the at least one labeled probe, specific for a portionof the designated genetic sequence, at the first and second locations onthe substrate, and comparing the signal from the first and secondlocations on the substrate to detect a designated genetic sequence inthe sample of tissue.

According to another aspect of the invention a method is provided todetect a designated genetic sequence in a sample of tissue by comparingthe sample with a designated control sample of tissue. This methodcomprises the steps of: treating the sample of tissue and the designatedcontrol sample of tissue with a sufficient amount of a lysis buffer toobtain cellular debris including genomic DNA; separating the genomic DNAfrom the cellular debris for the sample of tissue and the designatedcontrol sample of tissue using magnetic particles; depositing genomicDNA from the sample at a first location on a substrate; depositinggenomic DNA from the designated control sample at a second location onthe substrate; adding at least one labeled probe specific for a portionof the designated genomic sequence to the first and second locations onthe substrate; detecting the signal from the at least one labeled probe,specific for a portion of the designated genetic sequence at the firstand second locations on the substrate, and comparing the signal from thefirst and second locations on the substrate to detect the designatedgenetic sequence in the sample of tissue.

According to another aspect of the invention a method is provided todetect a designated genetic sequence in a sample of tissue by comparingsaid sample with a designated control sample of tissue. This methodcomprises the steps of: treating the sample of tissue and the designatedcontrol sample of tissue with a sufficient amount of a lysis buffer toobtain cellular debris including genomic DNA; separating the genomic DNAfrom the cellular debris for the sample of tissue and the designatedcontrol sample of tissue using magnetic particles; adjusting genomic DNAconcentration to facilitate detection of the designated geneticsequence; depositing genomic DNA from the sample at a first location onthe substrate; depositing genomic DNA from the designated control sampleat a second location on the substrate; adding at least one labeled probespecific for a portion of the designated genomic sequence to the firstand second locations on said substrate; detecting the signal from the atleast one labeled probe, specific for a portion of the designatedgenetic sequence at the first and second locations on the substrate, andcomparing the signal from the first and second locations on thesubstrate to detect the designated genetic sequence in the sample oftissue.

In another aspect of the invention, a method is provided of screening asample for a designated genetic sequence by comparing said sample with adesignated control sample of tissue the screening method using at leastone labeled target-binding probe and at least one labeledrefererice-binding probe. This method involves the steps of: treatingsaid sample of tissue and the designated control sample of tissue with asufficient amount of a lysis buffer to obtain cellular debris includinggenomic DNA; separating the genomic DNA from the cellular debris for thesample of tissue and the designated control sample of tissue usingmagnetic particles; depositing genomic DNA from the sample at a firstlocation on a substrate; depositing genomic DNA from the designatedcontrol sample at a second location on the substrate; adding at leastone labeled probe specific for a portion of the designated geneticsequence to the first and second locations on the substrate; adding atleast one labeled reference-binding probe to the first and secondlocations on the substrate; detecting the signal from the at least onelabeled probe, specific for a portion of the designated genetic sequenceat the first and second locations on the substrate, detecting the signalfrom at least one labeled reference-binding probe at the first andsecond locations on the substrate; and comparing the signal from thefirst and second locations on the substrate to screen a sample for thedesignated genetic sequence.

According to another aspect of the invention a method is provided ofscreening genomic DNA for a designated genetic sequence, wherein atissue sample, including the genomic DNA, is sent by a remote user to ascreening laboratory. The method involves the steps of: facilitating theextraction of genomic DNA from a tissue sample by providing a lysisbuffer to a remote user; transmitting the tissue sample from the remoteuser to the screening laboratory; receiving the lysed tissue sample atthe screening laboratory from the remote user; separating the genomicDNA from the lysed tissue sample using magnetic particles; depositinggenomic DNA from the sample at a first location on a substrate location;depositing genomic DNA from the designated control sample at a secondlocation on the substrate; adding at least one labeled probe specificfor a portion of the designated genetic sequence to the first and secondlocations on the substrate; adding at least one labeledreference-binding probe to the first and second locations on thesubstrate; detecting the signal from the at least one labeled probe,specific for a portion of the designated genetic sequence at the firstand second locations on the substrate, detecting signal from at leastone labeled reference-binding probe to the first and second locations onthe substrate; and comparing the signal from the first and secondlocations on the substrate to screening a sample for a designatedgenetic sequence.

According to another aspect of the invention, a method is provided ofscreening a sample of tissue for a designated genetic sequence, bycomparing the sample with a designated control sample of tissue, whereinthe tissue samples, including the genomic DNA, are sent by a remote userto a screening laboratory. This method comprises the steps of:facilitating the extraction of genomic DNA from a tissue sample byproviding a lysis buffer to a remote user; treating the sample of tissueand the designated control sample of tissue with a sufficient amount ofthe lysis buffer to obtain cellular debris including genomic DNA;transmitting the tissue samples in the lysis buffer from the remote userto the screening laboratory; receiving the lysed tissue samples at thescreening laboratory from the remote user; separating the genomic DNAfrom the cellular debris for the sample of tissue and the designatedcontrol sample of tissue using magnetic particles; depositing genomicDNA from the sample at a first location on a substrate; depositinggenomic DNA from the designated control sample at a second location onthe substrate; adding at least one labeled probe specific for a portionof the designated genomic sequence to the first and second locations onthe substrate; detecting the signal from the at least one labeled probe,specific for a portion of the designated genetic sequence at the firstand second locations on the substrate, and comparing the signal from thefirst and second locations on the substrate to screen the tissue samplefor the designated genetic sequence.

According to another aspect of the invention, a method is provided ofscreening genomic DNA, in at least one sample, sent by a remote user toa screening laboratory, for a designated genomic DNA sequence, theremote user providing screening parameters via an electroniccommunications link to the screening laboratory and a supplier. Thismethod comprises the steps of: transmitting an access request from aremote user to a screening laboratory via an electronic communicationslink; transmitting an access enabling response from the screeninglaboratory to the remote user via an electronic communications link, theaccess enabling response including the screening parameters; selectingscreening parameters by the remote user; transmitting the selectedscreening parameter selections from the remote user to the screeninglaboratory via an electronic communications link; receiving screeningparameter selections from the remote user by the screening laboratoryvia said communications link; transmitting a request from the remoteuser via an electronic communications link to a supplier to obtainprobes conforming to selected screening parameters; receiving the probesby said laboratory; transmitting the sample from the remote user to thescreening laboratory; conducting screening of the sample, according tothe selected screening parameters, to obtain data; and transmitting thedata to the remote user via an electronic communications link.

According to another aspect of the invention, a method is provided ofscreening genomic DNA, in at least one sample, sent by a remote user toa screening laboratory for a designated genomic DNA sequence, the remoteuser providing screening parameters via an electronic communicationslink to the screening laboratory. This method comprises transmitting anaccess request from a remote user to a screening laboratory via anelectronic communications link, transmitting an access enabling responsefrom the screening laboratory to the remote user via an electroniccommunications link, the access enabling response including thescreening parameters; selecting screening parameters by the remote user;transmitting the selected screening parameter selections from the remoteuser to the screening laboratory via an electronic communications link;receiving screening parameter selections from the remote user by thescreening laboratory via the communications link; transmitting a requestfrom the screening laboratory via an electronic communications link to asupplier to obtain probes conforming to selected screening parameters;receiving the probes by the screening laboratory, transmitting thesample from the remote user to the screening laboratory, conductingscreening of the sample, according to the selected screening parameters,to obtain data; and transmitting the data to the remote user via anelectronic communications link.

According to another aspect of this invention, a method is provided ofscreening genetic DNA, in at least one sample, sent by a remote user toa screening laboratory for a designated genomic DNA sequence, the remoteuser providing screening parameters via an electronic communicationslink to the screening laboratory. This method comprises: transmitting anaccess request from a remote user to a screening laboratory via anelectronic communications link; transmitting an access enabling responsefrom the screening laboratory to the remote user via an electroniccommunications link, the access enabling response including thescreening parameters; selecting screening parameters by the remote user;transmitting the selected screening parameter selections from the remoteuser to the screening laboratory via an electronic communications link;receiving selected screening parameter selections from the remote userby the screening laboratory via the communications link; transmitting arequest from the screening laboratory via an electronic communicationslink to a supplier to obtain probes conforming to selected screeningparameters; receiving the probes by the screening laboratory;transmitting a sample of tissue in a lysis buffer from the remote userto the screening laboratory, the lysis buffer formulated to lysis thetissue in the sample during transit time between the remote user and thescreening laboratory; conducting screening of the sample, according tothe selected screening parameters, to obtain data; and transmitting thedata to the remote user via an electronic communications link.

According to another aspect of the invention, an automated apparatus forhigh volume screening and targeted mutagenesis screening of tissuesamples sent by a remote user to a screening laboratory is provided.This apparatus comprises means for transmitting an access request from aremote user to a screening laboratory via an electronic communicationslink; means for transmitting an access enabling response from thescreening laboratory, to the remote user via an electroniccommunications link with screening parameters; means for transmittingscreening parameter selections from the remote user to the screeninglaboratory; means for transmitting the sample from the remote user tothe screening laboratory; means for isolating genomic DNA from thesample; means for depositing the genomic DNA on a substrate; means forscreening genomic DNA; and means for transmitting the data to the remoteuser.

According to another aspect of this invention, a high volume apparatusfor screening a tissue sample for modified or mutated genomic DNAaccording to screening parameter selections made by a remote user isprovided. This apparatus includes an automated accessioning station forremoving liquid from a first well plate to a second well plate; anisolation station for isolating genomic DNA in the second well plates;an optical standardization station for adjusting DNA concentration inthe second well plate; an arraying station for depositing the genomicDNA from the second testing well plate on to a substrate; ahybridization station for hybridizing labeled probes that bind to theportions of the genomic DNA; a detection station for detecting the boundlabeled probes; means for making screening parameter selections by aremote user, the remote user communicating with the apparatus through anelectronic communications link; and means for communicating screeningresults to the remote user through an electronic communications link.

According to another aspect of the invention, a system of screeninggenomic DNA in a sample for a designated genomic DNA sequence isprovided. The system includes a computer having a processor, memory andweb browser wherein the computer is adapted to receive the screeningparameter selections from a remote user; and a workstation that analyzessamples of genomic DNA for the screening parameter selections whereinthe workstation includes a microarray imager.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the invention and its advantages willbe apparent from the following Description of the PreferredEmbodiment(s) taken in conjunction with the accompanying drawings,wherein:

FIG. 1 is an illustrative overview of the remote automated testingprocedures of the present invention.

FIG. 2 is a block diagram of one embodiment of the system.

FIG. 3 is a block diagram of the ordering procedure.

FIG. 4 is a block diagram of account registration.

FIG. 5 is a block diagram of survey of work.

FIG. 6 is an illustration of orientation for sample identification anddesignation.

FIG. 7A is a block diagram of the laboratory process system.

FIG. 7B is a block diagram of the laboratory process system.

FIG. 7C is a block diagram of the laboratory process system.

FIG. 7D is a block diagram of the laboratory process system.

FIG. 8 is a block diagram of standard laboratory stations.

FIG. 9 is an illustration of a heating cassette.

FIG. 10 is a screen display illustrating a document on the transgenicscreening laboratory's web site relating to an outcome file.

FIG. 11 is a graphical representation of the results.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method and system for high volumetransgenic and targeted mutagenesis screening. This invention provides amethod for rapid identification of an organism, whose genome possessesspecific genetic sequences that exist endogenously or has been modified,mutated or genetically engineered. All patents, patent applications andarticles discussed or referred to in this specification are herebyincorporated by reference.

1. Definitions:

The following terms and acronyms are used throughout the detaileddescription.

-   -   complementary—chemical affinity between nitrogenous bases as a        result of hydrogen bonding. Responsible for the base pairing        between nucleic acid strands. Klug, W. S. and        Cummings, M. R. (1997) Concepts of Genetics, fifth ed.,        Prentice-Hall, Upper Saddle River, N.J. (hereby incorporated by        reference)    -   copy number—the number of transgenes that have randomly        integrated into the genome.    -   deletion mutation—a mutation caused by the removal of one or        more nucleotides from a gene or chromosome.    -   designated genetic sequence—includes a transgenic insert, a        selectable marker, recombinant site or any gene or gene segment.

DNA (deoxyribonucleic acid)—The molecule that encodes geneticinformation. DNA is a double-stranded molecule held together by weakbonds between base pairs of nucleotides. The four nucleotides in DNAcontain the bases: adenine (A), guanine (G) cytosine (C), and thymine(T). In nature, base pairs form only between A and T and between G andC; thus the base sequence of each single strand can be deduced from thatof its partner.

-   -   electroporation—the exposure of cells to rapid pulses of        high-voltage current which renders the plasma membrane of the        cells permeable and thus allowing transfection.    -   embryonic stem cells (ES cells)—a cell of the early embryo that        can replicate indefinitely and which can differentiate into        other cells; stem cells serve as a continuous source of new        cells.    -   genome—all the genetic material in the chromosomes of a        particular organism;    -   its size is generally given as its total number of base pairs.    -   genomic DNA—all of the genetic information encoded in a cell.        Lehninger, A. L., Nelson, D. L. Cox, M. M. (1993) Principles of        Biochemistry, second ed., Worth Publishers, New York, N.Y.        (hereby incorporated by reference)    -   genotype—genetic constitution of an individual cell or organism.    -   germ-line—unmodified genetic material transmitted to progeny via        gametes.    -   gene targeting—the creation of a null or mutant allele by        homologous recombination or gene replacement.    -   heating cassette—housing mechanism for glass substrates while        heating    -   imaging cassette—housing mechanism for glass substrate while        imaging    -   inducible gene targeting—a method of gene targeting that allows        the inducible inactivation (or activation) of a targeted gene by        experimental manipulation, such as administration of a drug.        Example: Cre recombinase is a site-specific recombinase that        catalyzes the excision of DNA flanked by lox recognition        sequences. Since the promoter for Cre expression is sensitive to        the drug interferon, targeted deletion is inducible.

Internet—a collection of interconnected (public and/or private) networksthat are linked together by a set of standard protocols to form aglobal, distributed network. The World Wide Web (hereinafter web) refersto both a distributed collection of interlinked, user viewable hypertextdocuments (commonly referred to as web pages) that are accessible viathe Internet and the user and server software components which provideuser access to such documents using standard Internet protocols.

-   -   line—A line is a colony bred for a genetic condition.    -   lipofection—the introduction of transgenes across cell membranes        by using liposome vesicles formed by phagocytosis. This method        is advantageous in that it is tissue-specific.    -   microarray imager—is a reader used to detect luminescence from        samples bound or affixed to an optically flat substrate.    -   microarray technology—is a hybridization-based process that        allows simultaneous quantitation of many nucleic acid species,        has been described (M. Schena, D. Shalon, R. W. Davis, and P. O.        Brown, “Quantitative Monitoring of Gene Expression Patterns with        a Complementary DNA Microarray,” Science, 270(5235), 467-70,        1995; J. DeRisi, L. Penland, P. O. Brown, M. L. Bittner, P. S.        Meltzer, M. Ray, Y, Chen, Y. A. Su, and J. M. Trent, “Use of a        cDNA Microarray to Analyze Gene Expression Patterns in Human        Cancer,” Nature Genetics, 14(4), 457-60 (“DeRisi”), 1996; M.        Schena, D. Shalon, R. Heller, A Chai, P. O. Brown, and R. W.        Davis, “Parallel Human Genome Analysis: Microarray-Based        Expression Monitoring of 1000 Genes,” Proc. Natl. Acad. Sci.        USA., 93(20), 10614-9, 1996; hereby incorporated by reference.        This technique combines robotic spotting of small amounts of        individual, pure nucleic acids species on a glass surface,        hybridization to this array with multiple fluorescently labeled        nucleic acids, and detection and quantization of the resulting        fluor tagged hybrids with a scanning confocal microscope. This        technology was developed for studying gene expression.    -   microinjection—a technique for introducing a solution of DNA        into a blastocyst or pronucleus of a fertilized egg using a fine        microcapillary pipette.    -   mutation—a heritable change in DNA sequence resulting from        mutagens. Various types of mutations including frame-shift        mutations, missense mutations, and nonsense mutations.    -   null mutation—completely eliminates the function of a gene,        usually because it has been physically deleted.    -   recombination—The process by which offspring derive a        combination of genes different from that of either parent. In        higher organisms, this can occur by crossing over.    -   recombinant DNA—A combination of DNA molecules of different        origin that are joined using recombinant DNA technologies.    -   retroviral infection—retroviral vectors with recombinant DNA        incorporate their genome into the chromosomes of cells it        infects.    -   selectable marker—an approach to facilitate the detection of        targeted cells by decreasing the detection of random integrants        rather than increasing targeting efficiency. There are two types        of selectable genes: designated and negative. A designated        selector gene, such as neomycin, confers resistance to drugs        normally lethal to the cell. Cells that have incorporated        neomycin into their genome by homologous recombination will be        resistant to the drug neomycin. Conversely, non-homologous        recombination events will retain the negative selector gene. The        negative selector gene, such as HSV-tk, confers sensitivity to        certain drugs (cells expressing HSV-tk are sensitive to        gancyclovir) resulting in cell death. A selectable marker is a        genetic sequence.    -   site specific recombinase—an enzyme that promotes recombination        between specific DNA sequences.    -   secondary well plate—plate DNA is printed from.    -   source well plate—The plate that remote user fills with sample        and lypholized reagent.    -   targeted deletion—technique for inactivating a gene by deleting        it from the genome. May be accomplished by homologous        recombination or inducible gene targeting.    -   targeted mutagenesis—alteration of the germline by the        introduction of a site-directed mutation.    -   transfection—the uptake, incorporation, and expression of        recombinant DNA by eukaryotic cells.    -   transgene—the foreign gene or DNA.    -   transgenic—this term describes an organism that has had genes        from another organism put into its genome through recombinant        DNA techniques. These organisms are usually made by        microinjection of DNA in the pronucleus of fertilized eggs, with        the DNA integrating at random.    -   transgenic line—a transgenic mouse or organism strain in which        the transgene is stably integrated into the germline and        therefore inherited in Mendelian fashion by succeeding        generation.    -   web site—a computer system that serves informational content        over a network using the standard protocol of the World Wide        Web. A web site corresponds to a particular Internet domain name        such as TransnetYX.com.        2. Overview of the Systems Components and Operations:

The present invention provides a method and system for transgenic andtargeted mutagenesis screening. A system and method operating accordingto the features described herein can be used to screen about 2000samples per day, (using only an automated arrayer) or if fully automatedabout 100,000 samples per day. Additionally, a system and methodoperating according to the features described herein can providescreening results to a remote user 1 from the screening laboratory 20within 48 hours of receiving the screening parameter selections for aplurality of samples.

FIGS. 1-3 present an overview of certain features of the presentinvention. The present invention allows a remote user 1 with access to acomputer 5 to order transgenic and targeted mutagenesis screening ofsamples they submit to the transgenic or targeted mutagenesis screeningwebsite 19, hereinafter screening laboratory. Using the Internet orother communication link 7, the remote user 1 sends an access request 7from the remote user's computer 5 to a screening laboratory computer 9via an electronic communication link 7, such as the Internet. Thescreening laboratory website 16 will transmit an access enablingresponse to the remote user 1 via an electronic communication link, suchas the Internet. This response includes three distinct sections. Thethree sections are Account Registration 21, Survey of Work 23 and SampleIdentification and Designation 25.

Now referring to FIG. 2, a remote user 1 can access screeninglaboratory's website 19 via a communication link 7. The website 19 canbe housed by an order manager 22 such as Dotlogix® (Memphis, Tenn.). Anorder manager is a software ordering management system. In the preferredembodiment the software is the order management system developed bySpaceWorks, Inc. of Rockville, Md., and now provided by ManugisticsGroup, Inc., of Rockville, Md. The order manager 22 functions to managethe placement of the order and houses the web site 19. The orderreceived from the remote user 1 as recorded in the website 19, isreported to order manager 22, which is in electronic communication 7with the screening laboratory computer 9. The screening laboratorycomputer 9 includes LIMS 24, which is communicatively coupled to aprocess controller 26.

LIMS 24 is the generic name for laboratory information management systemsoftware. The function of LIMS 24 is to be a repository for data,control automation of a laboratory, track samples, chart work flow, andprovide electronic data capture. LIMS 24 can also, in anotherembodiment, be in direct communication with the remote user 1 via anelectronic communications link 7. Any standard laboratory informationsystem software can be used to provide these functions. In the preferredembodiment, the Nautilus® program (Thermo LabSystems, a business ofThermo Electron Corporation, Beverly, Mass.) is used.

The process controller 26 is communicatively coupled to the workstation14. The process controller provides commands to any portions of theworkstation 14 that are amenable to automation. See, e.g. Layne et al.,U.S. Pat. No. 5,968,731 (hereby incorporated by reference). For example,the process controller 26 directs the delivery of the probes to thesubstrate 229 in the hybridization station 96. The workstation 14 iscommunicatively linked 28 to LIMS 24. In this way, the workstation 14can provide data to LIMS 24 for the formulation of the outcome report249, via an electronic communication link 7, such as the Internet, tothe order manager 22 or remote user 1. In an alternative embodiment, theremote user 1 can be linked 7 to the screening laboratory 20 by a directphone line, cable or satellite connection.

Now referring to FIG. 4, the user's Account Registration section 21requires upon receiving access to the screening laboratory's web site, aremote user 1 accesses an existing account by entering an account number31. The user will then enter a password. The user is asked whether theuser is the primary user 33 or another authorized user 35. If a validpassword is entered, the user can place a new order 39. Alternatively,the user can check an order status 41 by providing an order number 43and can proceed to tracking 45. Alternatively, a new account 47 can byopened by providing institution name, principal investigator, address,phone number, fax number, electronic mail address, billing information,other authorized user names 49. A password is selected 51, confirmed 53and billing information 55 is provided by the user.

The Survey of Work section 23 has a drop down section that allows a userto make screening parameter selections. Now referring to FIG. 5, theseselections include designating if the samples are transgenic 60 ortargeted mutations 70.

A transgenic organism has genes from another organism put into itsgenome through recombinant DNA techniques. These animals are usuallymade by microinjection of DNA into the pronucleus of fertilized eggs,with the DNA integrating at random. The number of copies of thetransgene that integrates into the genome is uncontrollable. Atransgenic line refers an organism strain in which the transgene isstably integrated into the germ-line and therefore inherited inMendelian fashion by succeeding generations. A transgene is any foreignDNA sequence or gene.

In the preferred embodiment, mice, i.e. the Genus Mus, are screened fortransgenic and targeted mutations. Some of the probes designated in theSurvey of Work section 23 are derived from Mus. Additionally, a geneticsequence present in all members of a species is used by the screeninglaboratory 20 as screening reference. For example, in the Genus Mus, themajor urinary protein MUP can be a reference genetic sequence. Hogan,B., Beddington, R., Constantini, F. and Lacy, E. (1994) Manipulating theMouse Embryo, second ed. Cold Springs Harbor Laboratory Press, ColdSprings Harbor, N.Y. (hereby incorporated by reference.)

All species of Mus can be screened with this method. Additionally, it isanticipated that other species can be screened according to the presentmethods. It is well within the ability of one skilled in the relevantart to make screening parameter selections for a different species andfor the screening laboratory to select a reference genetic sequence fora different genus species.

If the samples are transgenic 60, the remote user 1 is asked todesignate the genetic sequence 61, i.e. the transgenic insert, thenumber of lines to be tested, the number of samples per line 62, and thetarget genetic sequence to be targeted per line 63. The target geneticsequence is a portion of the designated genetic sequence and itcorresponds to the sequence of the probe. The remote user 1 is asked toidentify the probe sequence that is needed to be used for screening(usually 17 to 30 base pairs) per line 64, which is complementary to aportion of the designated genetic sequence. It should be noted thatwherever the term “screening” is used, these processes also refer to“detecting”. The probe sequence is complementary to the target geneticsequence. The remote user 1 identifies a probe sequence 64 that willhybridize, i.e. bind the target genetic sequence 63, if it is present inthe sample. This probe sequence is then communicated to a supplier andthe target-binding probe made by the probe provider will include thissequence.

The remote user 1 is then asked to identify characterizations 65 aboutthe designated control(s) provided by the remote user 1. The designatedcontrol is a genomic DNA sample known to have the designated geneticsequence. The designated control is submitted by the remote user 1 tothe screening laboratory 20. Additionally, the remote user 1 providescertain characterizations known about the designated control, includingidentifying the zygosity, copy number and the mosaic nature of thedesignated control. The unknown samples copy number can be extrapolatedand may accompany the quantitative results relative to the designatedcontrol sample.

With respect to targeted mutagenesis screening 70, the remote user 1 isasked to identify the number of lines and samples 71. The remote user 1is asked if the genetic modification is a knock-out or knock-in 72. Ifthe remote user 1 designated that a selectable marker is present 73,then a choice of marker will be presented to the user 74. The selectablemarker sequence is the designated genetic sequence. Common selectablemarkers include, but are not limited to, the genetic sequence forneomycin resistance, hygromycin resistance and puromycin resistance.Once the remote user 1 identifies which selectable marker is present,the genetic sequence is then presented to the user 1. Neomycin SequenceSEQ ID NO:1) ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACGTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCGGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTT GACGAGTTCTTCTGAHygromycin Sequence (SEQ ID NO:2)ATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGGTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAG Puromycin Sequence (SEQ ID NO:3)ATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCCGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGTGCCCGAAGGACCGCGCGACCTGGTGCATGACCCGCAAGCCCGGTGCCTGA

The remote user 1 is asked to review the sequence base-by-base andconfirm that the sequence presented is indeed present in their sample74. A portion of the selectable marker is designated as the targetgenetic sequence 63. The probe sequence is designated 64. The probesequence 64 binds to the target genetic sequence 63.

If the remote user 1 indicates that the selectable marker has beenremoved or that the sample has undergone site-directed recombinantmutations 75. The remote user 1 is directed to indicate whichrecombinant technology was employed to mutate their samples. The remoteuser 1 is presented with common recombinant technologies, which mayinclude, but are not limited to Cre-lox 78 and yeast FLP/FRT 79. Afterselecting one of the techniques a sequence such as (SEQ ID NO:4)ATAACTTCGTATA ATGTATGC TATACGAAGTTAT and (SEQ ID NO:5) GAAGTTCCTATACTTTCTAGA GAATAGGAACTTC C GAATAGGAACTTC CTTCAAGGATATG AAAGATCTCTTATCCTTGAAG G CTTATCCTTGAAGis presented to the remote user 1, which correlates to Lox-p site 78 andFRT site 79, respectively. The remote user 1 is asked to review thesequence base-by-base and confirm that the sequence presented is indeedpresent in their sample. The recombinant sites are the designatedgenetic sequences for selectable marker removal. The remote user 1designates a target genetic sequence that corresponds to a portion ofthe recombinant sequence 78 or 79. The remote user 1 identifies a probesequence 77 that will hybridize, i.e. bind the target genetic sequence63 if it is present in the sample. This probe sequence 77 is thencommunicated to a supplier and made according to the designation.

The remote user 1 is then asked to identify characterizations about thedesignated control(s) provided by the remote user 1. The designatedcontrol is a genomic DNA sample known to have the designated geneticsequence. The designated control is submitted by the remote user 1 tothe screening laboratory 20. Additionally, the remote user 1 providescertain characterizations known about the designated control, includingidentifying the zygosity, copy number and the mosaic nature of thedesignated control. The unknown samples copy number can be extrapolatedand may accompany the quantitative results relative to the designatedcontrol sample.

In the preferred embodiment, remote user 1 is asked to identify theirname, unique pre-registered account number, password and submit theirorder to the company. The insert for a transgenic sample or geneticsequence of the selectable marker for targeted mutagenesis screening canbe collectively referred to as the designated genetic sequence. Thetarget is any subset of the designated genetic sequence.

Now referring to FIG. 6, once the remote user 1 submits the Survey ofWork section the remote user 1 will be presented with the SampleIdentification and Designation section 25. The Sample Identification andDesignation section 25 includes 96 well plate locations. The remote user1 designates which sample was placed into each well. If the remote user1 has more than 96 samples, subsequent 96 source well plates anddesignations are available. With respect to FIG. 6, a 96 well platehaving a barcode accession number 3 will be shown oriented in thelongitudinal direction having X axis labeled H to A and Y axis labeled 1to 12 80. The X and Y axes designate a well position such as A1 81.

Now referring to FIG. 6, the remote user 1 is asked to provide: plateaccession number 82, number of lines 83, genetic line identification 84,number of samples 85, target sequence 86, probe sequence 87 and welllocation 88. The remote user 1 is then asked if the material depositedin well A1 is a control. If the material is a control 89, than theremote user designates the zygosity, mosaic nature and copy number ofthe material 90. If all of these parameters are not known, then theremote user enters as much information as is known. The remote user 1 isthen asked for any internal sample identification number 91.

Now referring to FIGS. 1 and 2, the remote user 1 transmits his or herorder including the completed screening parameter selection to thescreening laboratory 20 via a form of electronic communication 7 such asthe Internet or a direct line. The remote user 1 can transmit theselected screening parameter selections to the screening laboratory viaan electronic communications 7 link. This link 7 can be direct orindirect. In the indirect route, the screening parameters aretransmitted to web site 19, wherein order manager 22 provides LIMS 24with the screening parameter selections. In the preferred embodiment,the order generates two electronic messages, which will be sent todifferent locations. The first message is cross-referenced in LIMS 24with a list of stocked probes. If the probe designated by the user isnot stocked, an order message is sent to a supplier 16, such as acontracted probe provider. This request can be transmitted from remoteuser 1 to screening laboratory 20 via any form of electroniccommunication, and then via a form of electronic communication 10 tosuppliers' computer 8, or in the alternative, the order message can gofrom user 1 via any form of electronic communication 12 to suppliers'computer 8.

This supplier 11 creates the probe that the remote user 1 has designatedin their order for the screening the genomic DNA for the designatedgenomic sequence. The made to order probe can be referred to as thetarget-binding probe. This supplier 11 will then barcode and overnightship 13 the target-binding probe to the screening laboratory 20. Oncethe target-binding probes for each order for that days screening, isreceived by screening laboratory 20, the barcodes on the target-bindingprobes are scanned into LIMS 24. The LIMS 24 records the date and timethe target-binding probes were received along with the quality controldata provided from the probe provider.

In the preferred embodiment, the target-binding probes are placed on inworkstation 14 and LIMS 24 will record the barcode of the probe andrecord its specific location on the deck of the workstation 14, as willbe discussed in more detail with respect to the Hybridization Station96. Additionally, the screening laboratory 20 and the LIMS 24 systemcorrelates which target-binding probes will be used on which samples, aswill be discussed in more detail with regard to the HybridizationStation 96.

The second message, in the preferred embodiment, that is generated fromthe order placement of the remote user 1 will be to ensure the usershave the proper supplies to package and ship their samples. This messagewill define the number of well plate(s), shipping labels and amount ofreagents needed for the user. This request will be cross-referenced withan inventory list located in LIMS 24 at the remote user's location. Thisrequest can be sent from the remote user 1 to laboratory 20 via any formof electronic communication 7, and then via a form of electroniccommunication 10 to suppliers 11 or suppliers' computer 8, or in thealternative, the request can go from the remote user 1 via any form ofelectronic communication 12 to suppliers 11. If the appropriate amountof supplies are located within the user's facility a message will besent to the user defining the location where they can procure theshipping material needed. However, upon cross-referencing knowninventories if a sufficient number of supplies cannot be confirmed atthe user's location these items will then be packaged 18 and shipped tothe user 14. The remote user 1 will receive a message to inform themthat materials are being shipped to them with an expected time ofarrival.

Once the remote user 1 procures or receives these supplies, they placethe appropriate samples into the source well plates 2. The samples canbe obtained from prokaryotic or eukaryotic organisms. The samples may bea tissue sample from a mouse 8, but can also come from other animals andplants. In the preferred embodiment, mouse tails or ears are snipped toprovide a tissue sample. A source well plate 2 is a 96 well plate or thelike that receives the tissue sample and a sufficient amount of lysisbuffer to cover the tissue sample during transit to the screeninglaboratory 20. A source well plate 2 has an accession number 3 affixedto the side of the plate. The accession number is used by LIMS 24 totrack the source of well plate 2. The remote user 1 places theappropriate samples into the well locations in the source well plate 2that they had previously designated while placing their order FIG. 6.Once the samples are in the proper wells in the source well plate 2 thenthe remote user 1 dispenses a predetermined amount of reconstitutedlyophilized buffer 4 to cover the sample into each well using a pipette.The buffer is formulated to lysis the tissue to obtain cellular debrisincluding genomic DNA. More specifically, the buffer is formulated tolysis the sample while in transit between remote user 1 and thescreening laboratory 14. The transit time is approximately 24 hours asall samples are shipped via an express delivery service, such as FedEx®(Memphis, Tenn.). More specifically, for example, the buffer can be madeof (4M Urea, 0.1 M Tris-HCl (pH˜7.5), 1-mM NaCl, 10 mM EDTA, 1% SDS, 5mM DDT and 415 mg of proteinase K and RNase). The remote user 1 will addlysis buffer 4 to each well of the source well plate 2. The buffer 4should completely cover the samples. Once the samples and lysis bufferare in the source well plate 2 then a seal will be placed on the top ofthe source well plate 2 preventing samples from leaking. A plastic lidwill then be placed on the seal for transportation. The remote user 1will then place the source well plate 2 into an overnight deliveryservice package 15. The remote user 1 will then seal the package andship 16 to screening laboratory 20, and apply a barcode shipping label.

Now referring to FIG. 7A-D, the preferred embodiment of the presentinvention is shown. In FIG. 7A, the source well plates 2 arrive 101 atthe screening laboratory 20. The tracking number of the shipping labelis read with a barcode reader 103. If the shipping label is unreadable105, the tracking numbers are manually entered 107. The scanning of thetracking number is received 104 in LIMS 24 and a received message isposted to the user's account as shown in tracking field. The source wellplates 2 are removed from the package and taken to a clean room 109. Thesource well plates 2 contain the raw biological matter and lysis buffer.The source well plates 2 individual barcodes are scanned by the barcodereader 111 and recorded 106 in LIMS 24 as accession numbers. LIMS 24 cansend 106 a probe order to supplier 11 through the order manager 22. Ifthe source well plates 2 individual barcodes are unable to be scanned113, the accession numbers are entered manually 115. If the trackingnumber, accession number user order and worklist properly correlate,LIMS 24 will activate (not shown) an active record number for theplates.

The source well plates 2 are loaded 116 into a transportation apparatusin a clean room. A transportation apparatus is any device that holdswell plates and that can dock with the workstation. The transportationapparatus, in the preferred embodiment, includes several rigid traysstacked vertically in a housing unit that is mobile. This transportationapparatus can be moved between different automated stations, docked andthe rigid trays can be removed in an automated fashion and processed onthe deck of a workstation. Each rigid tray consists of nine locationsfor well plates. Each of these nine locations per tray has a uniquebarcode designating its specific location inside the transportationmodule.

The source well plate 2 accession number 3 is scanned with a barcodereader and the bar-coded well plate location in the transportationapparatus is scanned. The barcodes of the source plates 2 are married106 in LIMS 24 with the unique barcode locations in the transportationapparatus for tracking purposes. The source well plate 2 is physicallyplaced 117 into the transportation apparatus. LIMS 24 records andassociates 106 the well plate to this location. Once the transportationapparatus is loaded with the source well plates 2, the transportationapparatus is docked 119 into the workstation 14.

LIMS 24 will generate a worksheet for laboratory personnel (not shown).The worksheets outline the number of assay plates required and thevarious probes that will be needed. The LIMS 24 worklist will generate asingle file. The file format may include, but is not limited to, ASCII,XML or HTML. The file will be written into a specified directory on thenetwork drive. The name of the file will be unique and will correlate toa run number. The extension will be unique for worklist files.

We now refer to FIG. 8, a block diagram depicting one embodiment of theworkstation. Standard laboratory stations are logical groupings oflaboratory operations. These groupings, however, do not necessarilyrefer to different physical stations. These groupings include: AutomatedAccessioning Station 92, Isolation/Purification Station 93, OpticalStandardization Station 94, Arraying Station 95, Hybridization Station96 and Detection Station 97.

The following description provides the preferred embodiment, althoughone skilled in the art could elect to conduct these methods with varyingdegrees of automation as required.

3. Automated Accessioning Station 92

An Automated Accessioning Station 92 provides a device to remove liquidfrom the source well plate 2 to the primary master well plate. Theprimary master well plate is the plate in which the DNA is isolated. Anycommercially available automated accessioning device can perform thisfunction such as Genesis® Tecan (Raleigh-Durham, N.C.) or Multimeck®Beckman (Indianapolis, Ind.). These devices are referred to as liquidhandlers. The liquid handler delids the rigid plastic cover of thesource plate 121. In the preferred embodiment, liquid detection isperformed by the liquid handler by piercing the barrier sealingmechanism 123. The liquid handler performs liquid detection to verifythe existence of the original sample 125. The source well plates 2barcodes are re-scanned 127. This measurement will be recorded andposted 108 into the LIMS 24 database and reflected in the outcome report249. Additionally, LIMS 24 ensures 108 that well plates are consistentfrom transportation apparatus to the Automated Accessioning Station 92.Error codes will be generated if insufficient amount of raw testingmaterial is available. The liquid handler utilizes stainless steel, orthe like, pipette tips that are washed between each sample transfer.

The DNA is transferred 129 to clean well plates, referred to a primarymaster well plate. The barcodes of the primary master well plates arescanned 131 and LIMS 24 marries 102 to the new barcodes for the primarywell plates. The automated process accessioning continues until all ofthe days pending samples are accessioned into the primary master wellplates.

4. Isolation/Purification Station 93

The tray of primary matter well plates is moved by the transportationapparatus to the Isolation/Purification Station 93. In this station, thegenomic DNA will be isolated and purified using a separation method suchas magnetic or paramagnetic particles. The term “magnetic” in thepresent specification means both magnetic and paramagnetic. The magneticparticles can range from 0.1 micron in mean diameter to 100 microns inmean diameter. The magnetic particles can be functionalized as shown byHawkins, U.S. Pat. No. 5,705,628 at col. 3 (hereinafter '628 patenthereby incorporated by reference). In the preferred embodiment, themagnetic particles are 1 micron carboxylated iron core particles, butother magnetic particles with different functional groups of differentsize can be used.

For example, in the Isolation/Purification Station 93, each well of theprimary master well plate is filled with magnetic particles 133. Theparticles are dispensed into the well via a syringe pump. A secondsyringe pump dispenses a binding buffer into the wells containing theraw biological material and active particles 133. The dispensing itselfmay be sufficient to facilitate mixing of the samples. A secondarymixing mechanism, such as a tip can aspirate and redispense the liquid.A binding buffer, such as, 20% polethylene glycol (PEG) 8000, 0.02%sodium azide and 2.5M sodium chloride is used to non-specifically bindthe genomic DNA to the surface chemistry of the magnetic particles. ThePEG allows for hydrogen binding of water, which causes concentration ofthe DNA. Additional binding parameters are disclosed in Hawkins' 628patent. The particles, binding buffer and raw biological material areallowed to incubate at room temperature for ten minutes. Afterincubation, a magnet contacts the bottom of the primary master wellplates for several minutes, i.e. two to six minutes 137. The magneticparticles with attached genomic DNA are magnetically attracted to thebottom of the master well plates forming a pellet of particles. Thesupernatant is removed 139. A wash buffer, for example 70% ethanol and30% de-ionized water, is used to resuspend the particles 141. Themagnetic particles with the attached genomic DNA are separated from thesupernatant using a magnet 143. The supernatant is aspirated 145. Theparticle washing step is repeated two to four times.

The primary master well plates with pelletized particles are air dried147. In an alternative method, the pelletized particles can be driedwith compressed nitrogen. Once the particles are completely dry, themagnet is removed 149. The particles with attached genomic DNA areresuspended in a suspension buffer 151. A suspension buffer formulatedto elute the bound DNA from the particles. An example of one suchsuspension buffer is 0.01 M Tris (pH 7.4), 0.02% Sodium Azide or SodiumSaline citrate (SSC), dimethyl sulfoxide (DMSO), sucrose (20%) orforamide (100%). In the preferred embodiment, the primary master platesare heated 153 to 80° C. for two minutes to disassociate the DNA fromthe particles.

After heating and resuspending the DNA in solution, the magneticparticles are separated from the purified DNA using a magnet 155. Thesupernatant is removed 157 from the particles and is pipetted into asecondary well plate 2. The barcode of the secondary well plate is read.LIMS 24 will correlate the barcodes of the primary and secondary wellplates 114. A small amount (1-10 μl) of DNA supernatant is pipetted 159into a clean bar-coded optical 1536 well plate.

If a fully automated system is desired, the magnetic separator can beautomated and rise from the bottom of the workstation and make contactwith bottoms of all primary well plates simultaneously.

In one embodiment, the genomic DNA can be sonicated before or afterseparation with the magnetic particles 161. In the preferred embodiment,the genomic DNA is sonicated after separation from the cellular debris.Sonication can be done by any conventional means such as a fixed horninstrument. In the preferred embodiment, the genomic DNA is sonicatedfor 5 minutes to produce DNA fragments. Although there is a wide rangeof fragments from about 100 base pairs to up to 1 kilobase, the averagesize of the fragment is around about 500 base pairs (about meaning 50base pairs).

5. Optical Standardization and Well Plate Station 94

Optical Standardization involves DNA quantification. An optical plate,such as a 1536 well plate with a clear bottom from which an absorbentreading can be measured, is provided. In the preferred embodiment, a1536 ULTRAMARK (Bio-Rad, Hercules, Calif.) is used. The barcode of theoptical plate is scanned 161. Small aliquots of DNA supernatant from thesecondary master well plates are tracked 110 via LIMS 24 to specificwell locations within the DNA concentration optical well plate. Theoptical well plate is subjected to a DNA concentration analysis 163.This analyses involves an optical density scan (260/280 ratio) or afluorometry as known by one skilled in the art. The DNA concentrationvalues are quantified and recorded 112 in LIMS 24.

The concentration of genomic DNA in the secondary well is preferablyadjusted to be within the range of about 12.5 to 500 ng/μl of fluid inthe secondary master well plate and more preferable to be within therange from 17 ng to 250 ng/μl of fluid in the secondary master wellplate.

The optical standardization station 94 performs adjustments based onknown sample volumes in secondary master well plates with the known DNAconcentration to calculate the volume to hydrate or the time todesiccate each sample. The secondary well plate samples may be hydratedwith de-ionized water by the automated liquid handler system to decreasethe DNA concentration 165. Conversely, samples may be desiccated for acalculated time frame with compressed gas to concentrate the DNA samples167. If the DNA concentration is zero or the quantification value fallsbelow the parameters for optimization the LIMS 24 will generate aninsufficient quantity report to be noted on the outcome report (notshown). The optimized sample 169, in the secondary master well platesare re-scanned for concentration verification 171.

6. Arraving Station 95

In the Arraying Station 95, a sample of genomic DNA from the secondarywell plate is deposited on a substrate 229. A substrate is shown in FIG.9. A substrate 229 is optically flat so that it can be scanned with alaser and it includes a sufficient number of functional groups to bindthe genomic DNA to be screened. The substrates 229 may be glass,plastic, membranes, or a combination of the elements. Typically, thesubstrates 229 have some surface chemistry attached. These surfacechemistries include by not limited to amine groups, aldehydes groups orpolylysine. The reactive groups covalently or non-covalently attach thenucleic acid (DNA, cDNA, EST, Amplicon, etc.) to the surface of thesubstrate 229. In the preferred embodiment, aldehyde function groups(5.0×10¹²), reactive groups per cm are affixed to optically flat glassslide. The slide (SMA-1000) is purchased from TeleChem International,Inc., of Sunnyvale, Calif. (“Tele-Chem”).

In the Arraying Station 95, the genomic DNA is deposited 175 on thesurface of the substrate 229 with a solid pin tool using the automaticarrayer. An arrayer is a machine that dips titanium tips, or the like,into wells and prints on substrates. An automatic arrayer includessoftware that tracks the location of a specific sample with its locationon the substrate. The arrayer is communicatively coupled to LIMS 24 andinformation on each sample is transmitted 114 to LIMS. Typically,automatic arrayers include, but are not limited, to solid pin, splitpin/quill, tweezer, TeleChem's, pin and ring, piezoelectric technologyand syringe-solenoid technologies. An automatic arrayer can be used inthis method according to the manufactures operating instructions withoutmodification.

With the aldehyde-coated slides, the genomic DNA spots do not need to beprocessed further for attachment to the substrate. However, using otherfunctional groups, the genomic sample is attached on the substrates 229by ultra-violet cross-linking to the surface and/or thermally heating toattach the samples. For example, the genomic DNA is ultra-violetlyattached to the substrate at 1200 μ/j for thirty seconds. Similarly,heating at 80° C. for 2-4 hours will also accomplish the attachment. Thespots on the substrate 229 are from between 1-100 microns in size.Between approximately 1-130,000 genomic DNA spots, corresponding todiscrete trackable samples are located on an individual substrate 229.

Now referring to FIG. 7C, for example, the substrates barcode 231 isscanned 173. LIMS 24 associates 118 well plate and substrate barcodes231. Additionally, LIMS 24 associates 114 the substrate barcodes 231with a specific sample with a location on the substrate. Genomic DNAfrom the samples to be tested and genomic DNA from the designatedcontrol provided by the remote user 1 are deposited on the substrate inassigned locations, for example if referring only to the testing of onetissue sample, the first and second locations on the substrate. Prior todepositing the genomic DNA on the substrate, the genomic DNA samples aremixed with a sufficient amount of spotting buffer to facilitatedeposition on the substrate 229.

In the preferred embodiment, the spotting buffer is 3×SSC, but otherequivalent buffers may be used including DSMO, 1×SSC or commercialspotting buffers. The genomic DNA is deposited 175 on to the substrate229. The sample of genomic DNA is deposited three times on the substrate229 for quality control purposes. LIMS 24 records 114 the preciselocation of the deposited genomic DNA samples on the substrate 229 frominformation received from the automated microarray device. In analternative embodiment, at least one probe specific for a referencegenetic sequence is also added to each spot. The probe is specific forthe reference genetic sequence.

In another alternative embodiment, a small amount of morphology sequencenucleic acid is added 177 to each well of the secondary well plate. Themorphology sequence may be any nucleic acid sequence derived from anysource, such as prokaryotes or eukaryotes that does not naturally occurin the genome of biological material being tested. Lambda DNA spikedinto the sample could be used as a morphology control. Examples ofmorphology sequences may include but are not limited to exogenous genes,partial genes, tandem repeats, arbitrary sequences or syntheticoligonucleotides. The morphology sequence is pipetted into the genomicDNA of the secondary well plate and mixed by gently pipetting up anddown. The morphology control is used to determine if sample wassuccessfully transferred to the substrate 229.

Now referring to FIG. 7D, in the preferred embodiment, after thedepositing onto the substrate 229 is completed, the secondary wellplates are off loaded 197 from the Arraying Station 95. The secondarywell plate is sealed 199, the primary master plate is re-lidded 201 andthe barcodes of these plates are re-scanned and storage unit location isassigned 203. LIMS 24 marries the master plate to transportationapparatus location 204. The secondary well plates are then moved tofreezer 205. The secondary well plates that contain DNA samples, andoptionally the morphology control sequences, are sealed with a barrierseal. The barrier seal will prevent sample degradation and provide asafe storage mechanism. The transportation storage unit that houses thesecondary well plates after processing has a specific number as well asspecific locations within the unit. Each location inside the unit has anassociated unique barcode number. Each secondary well plate that isremoved from the workstation 14 will have it barcode scanned, as well asa scanning of the barcode of a specific location within thetransportation storage unit. LIMS 24 records the secondary well platenumber as well as its specific location. The marrying of the secondarywell plate with its location is useful if a sample needs to bere-accessed 204. The transportation storage unit will be moved to a coldstorage room for long-term storage.

7. Hybridization Station 96

The substrate 229 is placed in a heating cassette 177 for hybridization.Now referring to FIG. 9, a heating cassette 220 is shown, by way ofexample. This heating cassette 220 is made of a beveled top 225, aplurality of spacers 226, a metal frame 227 and tension clamps 230. Thesubstrate 229 is lowered into the metal frame 227 and plastic spacers226 are placed on top of the substrate 229 running lengthwise along theedge. The beveled top plate 225 is then lowered on around of thesubstrate 229 only separated by the plurality of spacers 226. The metaltension clamps 230 are then applied to the heating cassette 220, whichhold the cassette 220 together securely. The barcode of the substrate231 will extend beyond the heating cassette 220 to facilitate scanning.

Now referring to FIG. 7C, in the preferred embodiment, the heatingcassette 220 is assembled 178. The substrates 229 in the heatingcassettes 220 are transferred 179 to the heating block (GenePaint®—Tecan) (Raleigh-Durham, N.C.). The function of the heating blockis to increase and decrease temperature. In the preferred embodiment,the heating block is heated to 95-99° C. for two minutes in order toseparate the double stranded DNA making it more amenable tohybridization 181. The substrate 229 is then washed with 10 to 20volumes of ethanol 115. In the preferred embodiment, the substrate 229is then dried by forcing compressed N₂ into the top bevel of the heatingcassette forcing out any residual ethanol. A sufficient amount ofCasine, bovine serum albumine (BSA) or any commercial available blockingagent is dispensed 183 to the bevel of the heating cassette 220 to blockunbound surface chemistry, i.e. aldehydes. The heating cassette 230 isincubated 184 on the heating block. Following the blocking of thesurface chemistry with the blocking agent, the substrate 229 is washed185. In the preferred embodiment, the substrate 229 is washed withde-ionized water for one minute three different times.

The genomic DNA, which is immobilized on the substrate 229, ishybridized 187 with the probes. The LIMS 24 directs the HybridizationStation 96 to dispense reagents, such as probes, as selected by theremote user 1 in the Survey of Work 23. In the preferred embodiment,various probes are added to each DNA spot on the substrate 229. The spotcan be the sample to be tested or the spot can be the correspondingcontrol sample of DNA. The first probe is specific for a portion of thedesignated genetic sequence, which for both transgenic and targetedmutagenesis screening, is referred to as the target genetic sequence 63.The target probe specific for the target genetic sequence is referred toas a target-binding probe.

In the preferred embodiment, additionally, at least one second probespecific for a portion of the reference genetic sequence is added to thehybridization buffer. This probe can be referred to as areference-binding probe. The function of the reference-binding probe isto provide a designated quality control checkpoint. Thereference-binding probe has a genetic sequence that is complementary tothe gene or gene segment in the species being screened, i.e. thereference genetic sequence.

The endogenous gene used as a reference genetic sequence will have areiteration frequency similar to that of the transgene, so that thesimilar amount of hybridizations and linear curves will be obtained witheach probe. Individuals carrying 1-10 copies of a transgene, any singlecopy mouse gene can be used as the reference genetic sequence. Examples,of a single copy mouse gene present in the species Mus are shown in theTable 1. TABLE 1 (SEQ ID NO:6) 32.MMHAP9FLC5.seq.53FATCACAAGTACTGGGAGAGG (SEQ ID NO:7) MHAa67gl.seq.120FGTCTCAGAGGTTAACTCACC (SEQ ID NO:8) D9Mit211.1.38 TTCTTATCTTCAGCCCCACC(SEQ ID NO:9) X61434.129F ATAACACGGTGTGCACCACG (SEQ ID NO:10) U11075.95FTCCCTTCCTGTTGACTACAG (SEQ ID NO:11) Z49987.38F TACCCACACGGGCTTAAAAC (SEQID NO:12) 32.MMHAP9FLC5.seq.53R CACTGCCAGTGTGTTTTCAC

Additionally, for example, the mouse Major Urinary Protein gene familyinclude 20-30 copies per haploid genome. The gene sequences included inthe major protein family include: Mup_ctgtgacgtatgatggattcaataca. (SEQID NO: 13) mup tcggccatagagctccatcagctgga. (SEQ ID NO: 14) mupctgtatggataggaagggatgatgc. (SEQ ID NO: 15) mupggctcaggccattcttcattctcgggcct. (SEQ ID NO: 16)

To evaluate individuals that contain numerous numbers of transgeneintegration events, a probe for a ribosomal RNA gene works well.Ribosomal RNA gene can adequately elucidate 50 to several hundredcopies. Hogan, B., Beddington, R., Constantini, F. and Lacy, E. (1994)Manipulating the Mouse Embryo, 2^(nd) ed. Cold Springs Harbor LaboratoryPress, Cold Springs Harbor, N.Y. (hereby incorporated by reference.)

In addition to the target-binding probe, and the reference-bindingprobe, the morphology-binding probe can be added to the hybridizationbuffer. The function of the morphology-binding probe is to provide aquality control checkpoint to ensure that the printing process issuccessful. This quality control allows for the determination of whethera target sample was applied to the substrate. Also, it allows for theshape of the deposited sample to be evaluated in order to evaluatereproducibility across samples and substrates.

Once the target-binding probe, reference-binding probes, and optionallymorphology-binding probe are suspended in hybridization buffer the probeamplification molecules or secondary signal generation reagent is alsoadded to the mixture. The amplification molecules such as a dendriteprobe, has a nucleic acid capture sequence that is complementary to thetarget-binding probe, morphology-binding probe or reference-bindingprobes. Alternatively, the epitome of the target-binding probe,morphology-binding probe and reference-binding probes may be incubatedat 45° C. to 50° C. to pre-hybridize the probes with the secondarysignal generating reagent.

Different techniques may be employed in order to label the probes. Bothdirect labeling techniques and indirect labeling provide acceptableresults. The indirect methodology as is described in U.S. Pat. Nos.5,731,158; 5,583,001; 5,196,306 and 5,182,203 (hereby incorporated byreference). In the direct labeling technique, the labeled probehybridizes to the target genetic sequence. The probe will be directlymodified to contain at least one fluorescent, radioactive or stainingmolecule per probe, such as cyanine, horseradish peroxidase (HRP) or anyother fluorescent signal generation reagent. The fluorescent signalgeneration reagent includes, for example, FITC, DTAF and FAM. FAM is afluorescein bioconjugate made of carboxyfluorescein succinimidyl ester(e.g. 5-FAM (Molecular Probes, Eugene, Oreg.). DTAF is a fluoresceindichlorotriazine bioconjugate.

The indirect labeling techniques uses a probe that binds the selectedgenetic target sequence and that has been modified to contain aspecified epitome or if it has a nucleic acid binding sequence it formsa bipartite probe. The probes are made based on the remote user's 1screening parameter selections. The remote user 1 submits the probesequence 64 that correlates to the target genetic sequence 63. Inaddition to the target sequence 63, an additional binding sequencebeyond the specified target sequence 63 is added. The combination ofthese two elements gives rise to a bipartite probe.

For example, the binding sequence of the probe may have the samesequence as the 5′ end of reverse transcriptase. So the bipartite probewould contain the binding sequence of:

-   5′CCG GCT GAG TGA CGC GCA GAA GAC AGG GAC G—Probe Sequence 3′. (SEQ    ID NO:17). This binding sequence would then be complimentary to the    capture sequence for the Cy3 dendrite 5′ GGC CGA CTC ACT GCG CGT CTT    CTG TCC CGC C-3′ (SEQ ID NO:18).

The target genetic sequence 63 is specific for the probe geneticsequence 64 respectively. The binding sequence of the bipartite is freeand does not bind to the target genetic sequence 63. In the same manner,with respect to the reference genetic sequence, the reference-bindingprobe sequence is specific for the reference genetic sequence. Thebinding sequence of the bipartite probe is free and does not bind to theassociated genetic sequence.

Examples of bipartite probes, complementary to single copy mouse genes,are shown the table below: TABLE 2 (SEQ ID NO:10) AAA32.MMHAP9FLC5.5′-GGC CGA CTC ACT GCG CGT CTT CTG TCC CGC seq 53F CATCACAAGTACTGGGAGAGG(SEQ ID NO:20) AAAMHAa67gl.seq.120F 5′-GGC CGA CTC ACT GCG CGT CTT CTGTCC CGC CGTCTCAGAGGTTAACTCACC (SEQ ID NO:21) AAAD9Mit211.1.38 5′-GGC CGACTC ACT GCG CGT CTT CTG TCC CGC CTTCTTATCTTCAGCCCCACC (SEQ ID NO:22)AAAX61434.129F 5′-GGC CGA CTC ACT GCG CGT CTT CTG TCC CGCCATAACACGGTGTGCACCACG (SEQ ID NO:23) AAAU11075.95F 5′-GGC CGA CTC ACTGCG CGT CTT CTG TCC CGC CTCCCTTCCTGTTGACTACAG (SEQ ID NO:24)AAAZ49987.38F 5′-GGC CGA CTC ACT GCG CGT CTT CTG TCC CGCCTACCCACACGGGCTTAAAAC (SEQ ID NO:25) AAA32.MMHAP9FLC5. 5′-GGC CGA CTCACT GCG CGT CTT CTG TCC CGC seq.53R CCACTGCCAGTGTGTTTTCAC

Examples of bipartite probes, complementary to Mouse Major UrinaryProtein are shown in the table below. These bipartite probes arecomprised one of the MUP genetic sequences and a second genetic sequencethat is complementary to an amplification molecule. TABLE 3 MUP_probe 1(SEQ ID NO:26) 5′- GGCCGACTCACTGCGCGTCTTCTGTCCCGCCCTGTGACGTATGATGGATTCAATACA MUP probe 2 (SEQ ID NO:27)5′GGCCGACTCACTGCGCGTCTTCTGTCCCGCCTCGGCCATAGAGCTCCA TCAGCTGGA MUP probe 3(SEQ ID NO:28) 5′- GGCCGACTCACTGCGCGTCTTCTGTCCCGCCCTGTATGGATAGGAAGGGATGATGC MUP probe 4 (SEQ ID NO:29) 5′-GGCCGACTCACTGCGCGTCTTCTGTCCCGCCGGCTCAGGCCATTCTTCAT TCTCGGGCCT.

An amplification molecule, such as a dendrimer or tyramide, isintroduced. The amplification molecule is bound directly or indirectlyto the nucleic acid binding sequence or epitome. Free bipartite probesand excess amplification molecules are removed via several successivewash steps. The bound amplification molecule emits a signal that has alinear relationship to the number of bound molecule.

Typical modifications of binding probes include, but are not limited tobiotinylation and fluorescein attachments. A secondary signal generationreagents, such as an enzyme, is then bound to the epitome. The secondarysignal generation element may have a signal molecule directly attachedto it or it may activate or facilitate the attachment of another signalunit. Multiple signals units may be used to amplify the signal of thetarget.

Dendrimers, tyramide or the like are examples of amplification moleculesthat have traditionally been used to amplify cDNA for gene expressionanalysis and can be used in the present method.

In the preferred embodiment, LIMS 24 directs the hybridization station96 to direct a liquid dispenser to pipette the selected binding probesand the hybridization buffer. A number of hybridization buffers areacceptable, such as water and saline sodium citrate (SSC).Alternatively, buffer solutions such as 0.25 NaPO₄, 4.5% SDS, 1 mMEDTA,1×SSC or 40% Formamide, 4×SSC, 1% SDS may also be used.

The hybridization solution will then be applied 187 to the bevel top 225of the heating cassette 220. The substrates 229 in the heating cassette220 will be incubated 189. In the preferred embodiment, thehybridization mixture is incubated 189 for between 4 to 12 hours at atemperature ranging from 40° C. to 65° C. on the heating block after thetarget-binding probe, reference-binding probe and optionalmorphology-binding probe have hybridized to their respective targets. Itshould be noted that the hybridization solution can contain theamplification molecules or secondary signal reagents or they may beadded secondarily.

Once the substrates 229 have been incubated 189 with the hybridizationsolution the surface of the substrate is washed 191 several times toremove any excess reagent such as probe amplification molecules orsecondary signal reagents. In the preferred embodiment, the substrates229 will first be washed 191 and incubates at 55° C. with severalvolumes of 2×SSC, 0.2% SDS for ten minutes 189. The substrate will againbe washed at room temperature for 10 minutes with several volumes of2×SSC. The final wash will be conducted at room temperature for tenminutes with 0.2×SSC.

The substrate is dried 197 to facilitate imaging. In the preferredembodiment, the substrate is dried by forcing compressed Nitrogen intothe top bevel of the heating cassette. The compressed Nitrogen dryingwill continue for several minutes until all of the residual buffer isforced out of the heating cassette and the substrate is dry.

8. Detection Station 97

This detection station 97 involves detecting the signal from the atleast one labeled probe, specific for a portion of said designatedgenetic sequence at a first and second locations on said substrate, andcomparing the signal from the first and second locations on thesubstrate to detect a designated genetic sequence in the sample ofgenomic DNA.

In the preferred embodiment, the substrates 229 are then transferred tothe detection station 97. The substrates 229 are loaded into acommercially available imaging cassette, such as GSI Lumonics(Watertown, Mass.) and the imaging cassettes are loaded into themicroarray imager GSI Lumonics 5000 (Watertown, Mass.) used according tothe manufacturer's instructions. The substrate 229 is exposed to anexcitatory energy source to produce a quantifiable signal 195 from thesignal molecule. More particularly, the substrate's barcode will bescanned and reported 120 to LIMS 24. The substrate surface will bescanned with and at least three different channels and the results willbe recorded 120 in LIMS 24. The individual substrates 229 will havetheir barcodes scanned and married to a storage location barcode. Thereference-binding probes, the target-binding probe and optionally themorphology probe signal will be recorded and analyzed.

Now referring to FIG. 10, LIMS 24 now prepares the outcome report 249.Several calculations are performed in LIMS 24 before they are posted tothe outcome report 249. In the preferred embodiment, such calculationsinclude the evaluation of all three replicate per sample. The slope ofthe curve (quantified hybridization intensity/ng DNA) obtained with thetarget-binding probe is divided by the slope of the curve of thedesignated control probe for each individual sample. This is referred isthe induction ratio. The induction ratios are then compared to determinethe closeness of the replicates to the control and other replicates'induction ratios. Once the induction ratios are calculated, thequalitative and quantified results are posted to outcome reports 249.Calculating the linear relationship between the experimental quantifiedsignal and the quantified signals of designated control elucidates thecopy number, zygosity or mosaic nature of the sample. The ratio forhomozygous individuals should be twice the ratio of heterozygousindividuals. Additionally, the cell number is determined from the amountof genomic DNA that is recovered from the isolation process.

Now referring to FIG. 10, the sample outcome report 240 may includeaccount registration 250, well plate accession number(s) 252, controlsample locations 250 and genetic characterization of the designatedcontrol 252. Additionally, the outcome report 249 may include welllocation 254, sample identification 256, liquid level sensing 258, DNAconcentration 260, target sequence 262, probe sequence 264, signalquantification 266, qualitative results 268, zygosity/copy number 270,optical density reading per well before correction 274, optical densityreading after correction 276, estimated number of cells analyzed 278,quantitative analysis via comparison to designated control signalstrengths allowing for copy number estimation, zygosity or mosaic nature270. The outcome report 249 may also include a picture file (email) orpictorial representations of results 272. Additionally, informationgathered at the request of the remote user 1 from optimization andsequence confirmation quality control data and error messages will beincluded in the outcome report 249. The remote user 1 may choose to havethis file electronically sent or choose to be electronically notified.Additionally, remote user 1 has the option to have a hard copy sent viathe postal service.

Once the LIMS 24 has compiled all the data for the outcome report 249,the outcome report will be sent 7 to the remote user 1. In the preferredembodiment, LIMS 24 will send the report via a remote link 7 to eitherthe remote user 1 or the order manager 22, which can post the results onthe web site 16 or via an electronic link 7 send the outcome report 249.The LIMS 24 will keep results available for six months and then theresults will be recorded onto a long-term storage disk and archived.

In an alternative embodiment, high through put polymerase chain reaction(PCR) or variation of the PCR reaction such as Taqman® (PerkinElmer,Inc., Wellesley, Mass.) or molecular beacons sold by Integrated DNATechnologies, Inc. (Coralville, Iowa) can be used for conductingautomated transgenic and targeted mutagenesis screening. The user'saccount registration, survey of work and sample identification anddesignation could be created with the same or equivalent factors takeninto consideration. Additionally, the supplies, shipping tracking,sample tracking, quality control and results software architecture couldbe reproduced in a similar manner. The modification for the use of a PCRreaction would require the additional information being delineated inthe order process, specifically the survey of work. Using thisequivalent chemistry would require the user to designate criteria suchas the reaction buffer, magnesium concentration, primer sequences,primer concentration, dNTP concentration, cycle conditions and reactionvolume. The automated liquid handling could adjust these variables anddispense the reaction buffer to the appropriate location. While trackingthe samples through the automated system the genomic DNA may be isolatedin an automated fashion via a vacuum manifold isolation, microparticleisolation or chemical extraction. Isolated mammalian DNA can be loadedinto a high throughput thermocycler, such as the IAS's Genomatron(Boston, Mass.). Alternatively, substrates such as chemically treatedglass, plastic, membrane or any combination could be used as a reactionsubstrate. These substrates can be housed onto a heating block thatheats and cools according the thermal parameters of PCR. The liquidhandling platform would add the appropriate reaction buffer to eachsample of a well or across the surface of a substrate. The detection ofthe amplification can be staining via gel electrophoresis or capillaryelectrophoresis. Detection could also be qualified/quantified by theincorporation of fluorescent or radiolabeled dNTP's during the PCRreaction or via indirect staining methods.

An alternative PCR detection method would include the use of a Taqman®probe. The Taqman® probe is composed of a signal generating element(reporter dye) and a quenching element (quenching dye) that under normalconditions does not allow for the detection of the signal-generatingelement. The oligonucleotide Taqman® probe sequence is homologous to aninternal target sequence present in the PCR amplicon. The specifichybridization reaction of the Taqman® probe to the amplicon allows thequenching element to be separated from the signal-generating element.The signal molecule that is released is quantifiable. The signalstrength is proportional to the number of bound Taqman® primers.

Akin to the Taqman® technology is the molecular beacon technology.Molecular beacons can discriminate between single base variances. Theprobe is equipped with a signal generation element, such as afluorescent molecule, and a quencher element. Once the probe properlybinds to it complimentary sequence the quencher element is removed andthe fluorescent molecule is then detectable.

The following examples are provided by way of examples and are notintended to limit the scope of the invention.

9. EXAMPLES a. Example 1 Transgenic Screening

A remote user 1 accesses the web site 19 for the screening laboratory 20to order testing services. The remote user 1 enters the account number31 that had previously been specifically assigned by the screeninglaboratory 20. After the remote user 1 completes the AccountRegistration section 21 the remote user 1 is presented with the Surveyof Work section 23. The first designation that is required by the Surveyof Work section 23 by the remote user 1 is to identify if the samplesare of a transgenic or targeted mutagenesis in nature.

The remote user 1, in this example, designates that the samples to betested are of a transgenic 60 nature. The remote user 1 specifies 62only one transgenic line is to be tested. The designated geneticsequence 61 to be tested is “HUPPCA.” The remote user 1 then specifies47 transgenic samples needs to be screened from this line 62. The remoteuser 1 identifies the target genetic sequence to be detected 63. Theremote user 1 provides base sequence 64 of the probe is GCA AGG ACG CAAGGA AGC AGA G (SEQ ID NO: 30). That is complementary to the targetgenetic sequence 63. The probe sequence the user indicates is linked tothe binding sequence of: (SEQ ID NO:31) 5′-GGC CGA CTC ACT GCG CGT CTTCTG TCC CGC

Which results in the bipartite probe of: (SEQ ID NO:32) 5′-GGC CGA CTCACT GCG CGT CTT CTG TCC CGC GCA AGG ACG CAA GGA AGC AGA GThis binding sequence specifically couples to the capture arm of thereverse transcriptase sequence for the Cyanine 3 (Cy3) dendrimer.

The remote user 1 is presented with the Sample Identification andDesignation screen as shown in FIG. 6. The screen presents an image ofthe source 96 well plate in the proper orientation. This pictorialrepresentation aids the user in the process of specifically designatingeach sample into its correct Source 96 well location. Each of the remoteuser's samples are correlated, recorded and tracked to a specific wellof the source plate as shown in FIG. 6.

The remote user 1 loads the source well plate 2 with the proper samplesinto the correct locations as depicted in FIG. 6. Information about thenature of the designated control is ascertained. This informationincludes the zygosity and copy number, if known 90. The userreconstitutes the provided lyophilized lysis reagent with de-ionizedwater. The user pipettes a sufficient amount of lysis buffer 4 into eachwell of the source well plate 2 to cover the samples. The source wellplate 2 is then sealed with a barrier mechanism. To add additionalstructural integrity, the source well plate 2 is sealed with a rigidlid. The source well plate(s) 2 is loaded into the packing and shippingmaterials 17 provided. The package is transmitted 16 to the screeninglaboratory.

The package is received by the screening laboratory 20 and the sourcewell plate(s) 2 are removed and loaded into the workstation 14. Whileall samples are being individual tracked the samples have their genomicDNA extracted and optimized. The quantity of DNA is recorded. Thegenomic DNA sample and designated control sample are deposited on anoptically flat glass slide and hybridized with: Reference-bindingprobes: (SEQ ID NO:33) 5′-CCG GCT GAG TGA CGC GCA GAA TCA AGG GCGCTTCTTATCTTCAGCCCCACC

The 5′-CCG GCT GAG TGA CGC GCA GAA TCA AGG GCG element of the bipartiteprobe specifically binds to the unique capture arm of a Cyanine 5 (Cy5)dendrimer;

Target-binding probes:

5′-GGC CGA CTC ACT GCG CGT CTT CTG TCC CGC GCA AGG ACG CAA GGA AGC AGA G(SEQ ID NO: 32); and amplification molecules (Cy3 and Cy5 dendrimers).

An optically flat glass slide is subjected to an excitement energy laserand the quantifiable data is recorded. An outcome report 249 isgenerated and transmitted over the Internet 7 to the remote user 1. Theoutcome report 249 contains the account registration information 250,the well plate numbers 252, genetic characterizations 254, welllocations, sample identification 256, DNA concentration 260, targetsequences 262, probe sequence 264, signal quantification 266, results268, zygosity/copy number 270, estimated cell number 278, pictorialrepresentation 272, graphical representation 280, error messages 274 andquality control data 274.

b. Example 2 Targeted Mutagenesis

A remote user 1 accesses the web site 19 for the screening laboratory 20to order testing services. The remote user 1 enters the account number31 that had previously been specifically assigned by the screeninglaboratory 20. After the remote user 1 completes the AccountRegistration 21 the remote user 1 is presented with the Survey of Work23. The first designation that is required by the Survey of Worksection, by the remote user 1 is to identify if the samples are of atransgenic or targeted mutagenesis in nature.

The remote user 1 in this example designates that the samples to betested are of a targeted mutagenesis nature 70. The remote user 1specifies that only one 71 knock-out line 72, NSE-PPCA (neuron specificenolase), is to be tested. The designated genetic sequence, which is aknock-out line to be tested, is for the selectable marker hygromycin(SEQ ID NO:2). The remote user 1 then specifies 47 NSE-PPCA samples thatneed to be screened from this line. The remote user 1 identifies theselectable marker target sequence 73 of hygromycin to be detected 74.The remote user 1 specifically indicates that this selectable marker hasnot been removed 75 with recombinant technologies, such as Cre-10× orFLP/FRT. The selectable marker target genetic sequence is provided 63. Aprobe sequence for hygromycin is designated 64 (CAG GAT TTG GGC AAC ATCTT (SEQ ID NO:34)). The probe sequence 64 the remote user 1 indicates islinked to the binding sequence of: 5′-GGC CGA CTC ACT GCG CGT CTT CTGTCC CGC (SEQ ID NO:31).

This binding sequence specifically couples to the capture arm of thereverse ranscriptase sequence for the Cyanine 3 (Cy3) dendrimer.

The remote user 1 is presented with the Sample Identification andDesignation screen. The screen presents is an image of the source wellplate 2 in the proper orientation as shown in FIG. 6. This pictorialrepresentation aids the remote user 1 in the process of specificallydesignating each sample into its correct source well plate 2 location.Each of the remote user's 1 samples are correlated, recorded and trackedto a specific well of the source plate. The user identifies theaccession number 3 from the barcode of the source plate.

The remote user 1 loads the source well plate 2 with the proper samplesinto the correct locations as designated in FIG. 6 in the SampleIdentification section 25. Information about the nature of thedesignated control is ascertained 90. The remote user 1 reconstitutesthe provided lyophilized lysis reagent with de-ionized water. The userpipettes a sufficient amount of lysis buffer 4 into each well of thesource plate 2 to cover the samples. The source well plate 2 is thensealed with a barrier mechanism. To add additional structural integrity,the source well plate 2 is sealed with a rigid lid. The source wellplate(s) is loaded into the packing and shipping materials provided. Theremote user 1 places the shipping package 15 and ships 16 to thescreening laboratory 20.

The package 15 is received by the screening laboratory 20 and the sourcewell plate(s) 2 are removed and loaded into the workstation 14. Whileall samples are being individual tracked the samples have their genomicDNA extracted and optimized in an automated fashion. The quantity of DNAis recorded. The DNA is printed onto a substrate and hybridized withreference probes: (SEQ ID NO:35) 5′-CCG GCT GAG TGA CGC GCA GAA TCA AGGGCG CTTCTTATCTTCAGCCCCACC

The 5′-CCG GCT GAG TGA CGC GCA GAA TCA AGG GCG element of the bipartiteprobe specifically binds to the unique capture arm of a Cyanine 5 (Cy5)dendrimer, and target-binding probes: 5′-GGC CGA CTC ACT GCG CGT CTT CTG(SEQ ID NO: 36) TCC CGC CAG GAT TTG GGC AAC ATC TTand amplification molecules (Cy3 and Cy5 dendrimers).

An optically flat glass slide is subjected to an excitement energy laserand the quantifiable data is recorded. An outcome report 249 isgenerated and transmitted over the Internet 7 to the remote user 1. Theoutcome report 249 contains the account registration information 250,the well plate numbers 252, genetic characterizations 254, welllocations, sample identification 256, DNA concentration 260, targetsequences 262, probe sequence 264, signal quantification 266, results268, zygosity/copy number 270, estimated cell number 278, pictorialrepresentation 272, graphical representation 280, error messages 274 andquality control data 274.

c. Example 3 Eukaryotic Genomic DNA Magnetic Particle Isolation

Several mouse tails acquired from St. Jude Children's Hospital weretested. Samples were lysed, expelling the contents of the cell includingthe chromosomal DNA. The lysis buffer was made of 4M urea, 0.1 MTris-HCl (pH˜7.5), 180 mM NaCl, 10 mM EDTA, 1% SDS, 5 mM DDT, 415 mg ofProteinase K and Rnase. The samples were incubated in transport at roomtemperature for 24 hours. The lysate created a solution containinggenomic DNA without the cloned DNA elements from prokaryotic work. Thegenomic DNA was separated. The lysate was combined withcarboxyl-terminated iron oxide particles. Polyethylene Glycol (PEG)8000, 0.02% Sodium Azide, 2.5M NaCl was added and gently mixed. Thesamples were incubated for ten minutes at room temperature. The sampleswere exposed to a magnetic field for several minutes until thesupernatant cleared. The supernatant was aspirated and discarded. Themicroparticles were washed and re-suspended with 70% ethanol and 30%de-ionized water to remove any salt. The samples were exposed to themagnetic field until the supernatant was clear. The supernatant was thenaspirated and discarded. The ethanol washing was repeated. Themicroparticles were air dried for several minutes. The microparticleswere re-suspended in 0.01M Tris (pH 7.4), 0.02% Sodium Azide. TheTris-microparticle solution was incubated at 80° C. for several minutes.The samples were re-exposed to the magnetic field until the supernatantwas clear. The supernatant was aspirated and placed into clean tubes. Todetermine the recovery yield of genomic DNA, a PicoGreen quantificationassay was performed. PicoGreen® (Molecular Probes, Inc., Eugene, Oreg.)is a commercially available stain that binds only to double strandedDNA. Samples were loaded into cuvettes and were exited at 480 nm. Thefluorescence emission intensity was measured at 520 nm using aspectrofluorometer and plotted as a function of DNA concentration. TABLE4 Mouse Tail Tissue Genomic DNA 1A  450 ng 1B  430 ng 2  584 ng 3 2070ng 4 1944 ng

The results show that mammal genomic DNA in tissue or cells issuccessfully lysed at room temperature and genomic DNA is recovered withmagnetic particles.

d. Example 4 Immobilization Hybridization, Detection of MammalianGenomic DNA

To evaluate the difference and to determine the optimal conditions forwhich mouse genomic DNA can be bound to a substrate, hybridized to aprobe and have quantifiable signal detected several iterations wereconducted. There were two distinct types of mouse genomic DNA that wasunder study, sonicated and unsonicated. Both of these types of mousegenomic DNA were used to make serial dilutions in four differentbuffers. There were six serial dilutions of both types of mouse genomicDNA's made ranging from 538 ng/μL to 17 ng/μL. There were sevenendogenous gene sequences that are ubiquitous to all species of micethat were amplified with PCR to serve as designated controls.

Seven mouse markers that naturally occur in the mouse genome wereamplified using PCR. These markers function as controls for the unknownstock genomic DNA. The markers were: TABLE 5 (SEQ ID NO: 6)32.MMHAP9FLC5.seq.53F ATCACAAGTACTGGGAGAGG (SEQ ID NO: 7)MHAa67g1.seq.120F GTCTCAGAGGTTAACTCACC (SEQ ID NO: 8) D9Mit211.1.38TTCTTATCTTCAGCCCCACC (SEQ ID NO: 9) X61434.129F ATAACACGGTGTGCACCACG(SEQ ID NO: 10) U11075.95F TCCCTTCCTGTTGACTACAG (SEQ ID NO: 11)Z49987.38F TACCCACACGGGCTTAAAAC (SEQ ID NO: 12) 32.MMHAP9FLC5.seq.53RCACTGCCAGTGTGTTTTCAC

The PCR fragments were separated on an agarose gel and the ampliconswere isolated with using magnetic particles using the procedure set outin Hawkins '628 patent. A quantification analysis (using PicoGreen) wasemployed to determine the specific concentration of PCR amplicon DNA.Serial dilutions of the known concentrations of PCR Amplicon controlsand serial dilutions of both sonicated and un-sonicated stock endogenousmouse genomic DNA was printed onto the substrates. The amplicon (genericname for portion of genetic sequence that is amplified DNA and thegenomic DNA were fixed to the substrates via covalent and/ornon-covalent linkage. TABLE 6 Results from the quantification ofcontrols: gel well# lane PG5_raw PG5, ng/μl ng yield ug yield AmpliconA1 1 0 0.020434 A2 3 51.6 6.2 2976 2.98 U11075.95 A3 5 26.0 3.1 15021.50 q.120F A4 7 33.6 4.0 1940 1.94 D9Mi211.1.38 47.mmhap5flh A5 9 20.32.5 1177 1.18 5.seq.85 A6 11 28.5 3.4 1648 1.65 LC5.seq.53F A7 13 22.52.7 1305 1.31 Z49987.38F A8 15 11.2 1.4 655 0.65 X61434.129F A9 17 15.41.9 894 0.89 Actb-pAi. Results of the Control Genomic DNA Signal Serial Dilution:

PCR amplicons, printed onto substrates, probed, and detected are welldocumented in the literature. PCR amplicons are generally used for geneexpression work. These PCR amplicon controls were made into six serialdilutions in the four different buffers at a concentration gradient thatwas equivalent to the concentration gradient of the mouse genomic DNA's(538 ng/μL to 17 ng/μL). The sonicated and unsonicated mouse genomic DNAserial dilutions as well as the PCR amplicon control serial dilutionswere printed together onto six different types of substrates. Thesesubstrates were probed with two different types of probes: the FAMprobes (direct labeling) and the bipartite probe that was amplified witha dendrimer.

A well plate holding the serial dilutions was created as follows. Boththe sonicated and unsonicated eukaryotic and prokaryotic DNA wassuspended at different concentrations in 4 different buffers. The fourbuffers include 3×SSC, 50% DMSO, 5.5M NaSCN, and the fourth buffer isthe commercially available TeleChem printing buffer. The mouse genomicDNA was diluted in half 6 times (538 ng/μL, 269 ng/μL, 135 ng/μL, 68ng/μL, 34 ng/μL and 17 ng/μL). The PCR control serial dilutions werealso created. The control dilutions were made from the PCR-amplicons ofendogenous mouse genes that were previously amplified. The PCR controlswere generated as follows: the first four control serial dilutions weremade with 3×SSC buffer, 5.5M NaSCN, 50% DMSO and the TeleChemcommercially available buffer. The beginning concentration of the PCRamplicon DNA was 538 ng/μL. There were 6 dilutions of the PCR amplicons(538 ng/μL, 269 ng/μL, 135 ng/μL, 68 ng/μL, 34 ng/μL and 17 ng/μL). Thefifth and sixth controls were also diluted in a separate well plate witha starting amount of 538 ng/μL. Again they were suspended in 3×SSC, 5.5MNaSCN, 50% DMSO as well as the TeleChem commercially-available buffer.TABLE 7 384 Well Plate Design Mouse 3xSSC Mouse 3xSSC Control #1 3xSSCControl #1 5.5M NaSCN genomic 50% DMSO genomic 50% DMSO Control #2Control #2 sonicated 5.5M NaSCN sonicated 5.5M NaSCN Control #3 Control#3 TeleChem TeleChem Control #4 Control #4 Mouse 3xSSC Mouse 3xSSCControl #1 50% Control #1 TeleChem genomic 50% DMSO genomic 50% DMSOControl #2 DMSO Control #2 sonicated 5.5M NaSCN sonicated 5.5M NaSCNControl #3 Control #3 TeleChem TeleChem Control #4 Control #4 Mouse3xSSC Mouse 3xSSC Control #5 3xSSC Control #5 TeleChem genomic 50% DMSOgenomic 50% DMSO Control #6 Control #6 unsonicated 5.5M NaSCNunsonicated 5.5M NaSCN Control #7 Control #7 TeleChem TeleChem Control#8 Control #8

The following bipartite probes were used: TABLE 8 (SEQ ID NO: 19)AAA32.MMHAP9FLC5. 5′-GGC CGA CTC ACT GCG CGT CTT seq.53F CTG TCC CGCCATCACAAGTACTGGGAGAGG (SEQ ID NO: 20) AAAMHAa67g1.seq.120 5′-GGC CGA CTCACT GCG CGT CTT F CTG TCC CGC CGTCTCAGAGGTTAACTCACC (SEQ ID NO: 21)AAAD9Mit211.1.38 5′-GGC CGA CTC ACT GCG CGT CTT CTG TCC CGCCTTCTTATCTTCAGCCCCACC (SEQ ID NO: 22) AAAX61434.129F 5′-GGC CGA CTC ACTGCG CGT CTT CTG TCC CGC CATAACACGGTGTGCACCACG (SEQ ID NO: 23)AAAU11075.95F 5′-GGC CGA CTC ACT GCG CGT CTT CTG TCC CGCCTCCCTTCCTGTTGACTACAG (SEQ ID NO: 24) AAAZ49987.38F 5′-GGC CGA CTC ACTGCG CGT CTT CTG TCC CGC CTACCCACACGGGCTTAAAAC (SEQ ID NO: 25)AAA32.MMHAP9FLC5. 5′-GGC CGA CTC ACT GCG CGT CTT seq.53R CTG TCC CGCCCACTGCCAGTGTGTTTTCAC

The FAM modified probes (direct labeling) were used: TABLE 9 (SEQ ID NO:37) CCC32.MMHAP9FLC5.seq.53F ATCACAAGTACTGGGAGAGG (SEQ ID NO: 38)CCCMHAa67g1.seq.120F GTCTCAGAGGTTAACTCACC (SEQ ID NO: 39)CCD9Mit211.1.38 TTCTTATCTTCAGCCCCACC (SEQ ID NO: 40) CCX61434.129FATAACACGGTGTGCACCACGii. Sonication of Mouse Genomic DNA:

The mouse genomic DNA was sonicated for five minutes with a fixedsetting using a horn sonicator.

iii. Array Design:

The array design was as follows: there were six pallets, each holding 14slides for the array machine. Each pallet held one format of slides withthe exception of the right upper quadrant. Each pallet held one formatof slides. The left upper quadrant pallet held 14 superaldehyde slides.The middle upper quadrant pallet held 14 Schliecher Schuell (Dassel,Germany) slides. The right upper quadrant held 5 sigma slides as well as9 superamine slides. The lower left quadrant held 14 superamine slides.The middle lower quadrant held polylysine slides and the right lowerquadrant held the amino-sylinated slides (SCA slides).

The 384 well plate was loaded on to the array machine. The array machinewas calibrated to accurately deposit the correct amount of sample ontoeach substrate using a split-pin tip. The array machine plated thecontents of the 384 well plate across the 84 slides. This process tookapproximately 40 minutes to complete the entire printing. Afterprinting, the Fast Slides® from Schliecher Schuell (Dassel, Germany)were removed because the nitrocellulose membrane was damaged.

iv. Crosslinking the DNA:

These slides were UV cross-linked at 1200 μJ, with the exception of thealdehyde slides. All slides were boiled for 5 minutes to separate thedouble-stranded DNA on the surface of the substrate. The boiling of thesubstrates occurred in a slide holder and water. After the substrateswere boiled in water for 5 minutes, they were dunked into a 100% ethanolfor 1 minute, which facilitated the drying of the slides.

The FAM probes were re-suspended, making stock solutions of each probetype. 279 mM/279 L equals 1 μM. Each stock was 1 μM.

The direct-labeled FAM probes were all combined (multiplexed) into onehybridization buffer. This hybridization buffer was applied acrossdifferent substrates (1 μM of the probe or 0.3 μM in 30 μL). Thehybridization buffer was created as follows: Approx. 30 μL of solutionwere needed per substrate. The total volume of hybridization buffer was180 μL. This hybridization buffer includes 18 μL of 20×SSC, 0.2 μL ofprobe-one, 0.2 μL of probe-two, 0.2 μL of probe-three, 0.2 μL ofprobe-four, 1.2 μL of 10% SDS, 6 μL of BSA, and 154 μL of water,equaling a total of 180 μL of buffer. It should be noted FAMtraditionally has a high background and low signal. The FAMhybridization buffer was spun for seconds in a microfuge tube afterbeing vortexed for a few brief seconds to ensure proper mixing. The FAMcocktail was applied to the superamine slide 012320, the polylysineslide 012370, the sigma slide 012800, the superaldehyde substrate 012850and the CSA substrate of 012440. 30 μL of hybridization buffer wasapplied to the slides. The substrates with the hybridization buffer wasthen covered with a cover slip from Schliecher Schuell (Dassel, Germany)and were manipulated to remove any air bubbles from under the slidecover that could result in poor hybridization. The substrates wereplaced in the hybridization chamber with moistened towelettes to ensurethat the slides do not dry out. The chambers were placed in a 45.1° C.incubator for 30 minutes. Bovine serum albumin (BSA) was used toadequately bind the chemical groups on the substrate surface. After thehybridization process was completed the slides were washed in 2×SSC with1% SDS for approximately 5 minutes. The slides were washed for another 5minutes in 2×SSC. The slides were then washed in 0.6 SSC for 5 minutes,followed by a wash in 100% ethanol. The FAM slides were imaged seven dayafter probing and a detectable signal was quantifiable.

In the preferred embodiment, the bipartite probes as shown in Table 7were all combined (multiplexed) into one hybridization buffer. Thishybridization buffer was applied across the different substrates andincubated. The Cyanine 3 (Cy3) dendrimer was then used to bind to theprobe. Now referring to FIG. 11, the Y-axis 282 shows the intensity offluorescence of the labeled prove and the X-axis 284 is the sampleidentification slide. The data from one slide was used to create agraphical representation of the results shown as FIG. 11. The slideswere scanned and the quantifiable data was recorded. FIG. 11 shows thatgenomic DNA can be detected using a microarray imager.

More specifically, the results are shown in Table 10. TABLE 10 Sonicatedand Unsonicated Mouse Genomic DNA Quantified Results Relative to PCRControls Mouse PCR Genome Mouse Genome Control Un-Sonicated Sonicated #13xSSC 3xSSC PCR Control 3xSSC Cy3 Cy3 #5 3xSSC Cy3 Well Dens - WellDens - Well Cy3 Well Dens - Levels Inserted Levels Inserted LevelsInserted Dens - Levels Inserted 1608.88 A01 913.87 A13 12717 I01 1029.33I13 1829.92 A01 1418.65 A13 15406.2 I01 532.73 I13 10838.3 A02 1442.54A14 27048.1 I02 1165.44 I14 17623.8 A02 1683.92 A14 24489.3 I02 1499.83I14 1499.88 A03 971.92 A15 14616.8 I03 513.25 I15 1346.29 A03 781.81 A1519646.3 I03 868.75 I15 694.9 A04 637.83 A16 1207.77 I04 595.54 I161481.81 A04 496.44 A16 999.29 I04 468.33 I16 5819.92 A05 741.6 A179708.62 I05 738.31 I17 6805.9 A05 1056.92 A17 9224.71 I05 607.25 I171481.29 A06 719.58 A18 745.94 I06 1118.46 I18 1629.08 A06 1244.08 A181431.6 I06 891.85 I18 11949.7 A07 32821.2 A19 6173.75 I07 14934.1 I199454.98 A07 34354.4 A19 6054.54 I07 15255.2 I19 16005.6 A08 12853.5 A2023767.1 I08 33108.4 I20 21630 A08 4668.19 A20 17812.3 I08 31512.9 I2011463.3 A09 1513.67 A21 3185.6 I09 18818.8 I21 10900.2 A09 1343.58 A213039.54 I09 22902.9 I21 4657.04 A10 4713.79 A22 3636.42 I10 15394.6 I225670.4 A10 2990.83 A22 4685.83 I10 24232.1 I22 10906.7 A11 633.38 A238357.56 I11 1493.71 I23 9271.25 A11 805.77 A23 5246.52 I11 13973.6 I23713.48 A12 1075.42 A24 1381.31 I12 14037 I24 738.46 A12 904.88 A242326.31 I12 16366.9 I24

The results show that either sonicated or unsonicated genomic DNA canproduce a reportable signal when affixed to an optically flat substrateand read with a microarray imager.

Although the present invention has been described and illustrated withrespect to preferred embodiments and a preferred use thereof, it is notto be so limited since modifications and changes can be made thereinwhich are within the full scope of the invention.

1. A method of requesting and receiving a sample outcome report forgenomic DNA screening of a plurality of biological samples sent by aremote user to a screening laboratory, the remote user providingscreening parameters via an electronic communications link to thescreening laboratory, the method comprising: (a) transmitting an accessrequest from a remote user to a screening laboratory via an electroniccommunications link; (b) receiving an access enabling response from thescreening laboratory by the remote user via an electronic communicationslink, the access enabling response including the screening parameters;(c) selecting screening parameters by the remote user to provideselected screening parameter selections; (d) transmitting the selectedscreening parameter selections from the remote user to the screeninglaboratory via an electronic communications link; (e) transmitting aplurality of biological samples from the remote user to the screeninglaboratory wherein transmitting a plurality of biological samplesinvolves the step of placing one of each of the plurality of biologicalsamples in a corresponding well of a multi-well plate; and (f) receivinga sample outcome report for a plurality of biological samples from thescreening laboratory by the remote user via an electronic communicationslink, wherein the electronic communications link is the Internet.
 2. Themethod of claim 1 wherein the multi-well plate is a 96 well plate. 3.The method of claim 1 further comprising the step of correlating each ofthe plurality of biological samples with a well of the multi-well plate.4. The method of claim 1 wherein the step of receiving an accessenabling response includes a sample identification and designationsection.
 5. The method of claim 4 wherein the sample identification anddesignation section indicates a well plate having a plurality of wells,each well having an associated well position.
 6. The method of claim 4,wherein the sample identification and designation section presents anx-y orientation of a plurality of wells, the orientation defining aplurality of rows and a plurality of columns.
 7. The method of claim 6,further comprising the step of designating individual ones of the x-yorientation of a plurality of wells.
 8. The method of claim 1 whereinthe step of receiving an access enabling response includes a survey ofwork section for entering at least one screening parameter selection. 9.The method of claim 1 further comprising the step of designating each ofthe plurality of biological samples in the multi-well plate.
 10. Themethod of claim 1, further comprising the step of designating a controlsample of the plurality of biological samples.
 11. The method of claim1, further comprising the step of designating a plate accession numberof the multi-well plate.
 12. The method of claim 1 further comprisingthe step of designating which sample of the plurality of biologicalsamples is placed into each well of the multi-well plate.
 13. The methodof claim 1 wherein access request includes an account identifier and apassword.
 14. The method of claim 1 wherein the screening parameterselections include a selectable marker.
 15. The method of claim 1wherein the screening parameter selections include data indicative of anumber of lines to be tested.
 16. The method of claim 1 where thescreening parameter selections include a probe sequence.
 17. The methodof claim 1 wherein the screening parameter selections include adesignated control.
 18. The method of claim 1 wherein the screeningparameter selections include a designated genetic sequence.
 19. Themethod of claim 1 wherein the screening parameter selections include atarget genetic sequence.
 20. The method of claim 1 wherein the sampleoutcome report includes a pictorial representation of screening results.21. The method of claim 17 wherein the sample outcome report identifiesthe designated control.
 22. The method of claim 21 wherein the sampleoutcome report includes a quantitative analysis of each of the pluralityof biological samples in comparison to the designated control.
 23. Themethod of claim 21 wherein the sample outcome report includes aqualitative analysis of each of the plurality of biological samples incomparison to the designated control.
 24. The method of claim 1 whereinthe sample outcome report includes a well location of each of theplurality of biological samples in the multi-well plate.
 25. The methodof claim 1 wherein the sample outcome report includes a copy number foreach of the plurality of biological samples.
 26. The method of claim 1wherein the sample outcome report includes an accession number of thewell plate.
 27. The method of claim 1 further comprising the step ofelectronically showing the remote user a well plate having a pluralityof wells, each of the wells having a corresponding well plate locationon the multi-well plate.
 28. A computer-implemented method ofelectronically ordering transgenic or targeted mutagenic screening for aplurality of biological samples by electronic communications between awebsite and a remote user computer, the method comprising the steps of:(a) transmitting an account identifier and password from the remoteuser's computer to the website to gain access to the website; (b)designating at the remote user's computer a plurality of biologicalsamples for transgenic or targeted mutagenic screening; (c) designatingat the remote user's computer the location of the plurality ofbiological samples in the plurality of wells; and (d) designating agenetic sequence to be screened for each of the plurality of biologicalsamples.
 29. The method of claim 28 further comprising the step ofshowing on the remote user's computer a well plate having a plurality ofwells.
 30. The method of claim 28, further comprising the step ofproviding the number of the plurality of biological samples in theplurality of wells in the well plate.
 31. The method of claim 28,further comprising the step of designating a control sample.
 32. Themethod of claim 31 wherein the step of designating the control sampleincludes the step of identifying the zygosity of the control sample. 33.The method of claim 31 wherein the step of designating the controlsample includes the step of identifying a copy number of the controlsample.
 34. The method of claim 31 wherein the step of designating thecontrol sample includes the step of identifying mosaic nature of thecontrol sample.
 35. The method of claim 28, further comprising the stepof designating a probe sequence complimentary to a portion of thedesignated genetic sequence.
 36. The method of claim 28, furthercomprising the steps of: (a) transmitting results of the step ofdesignating the location of the plurality of biological samples in theplurality of wells to the website; (b) transmitting results of the stepof designating the genetic sequence of the plurality of biologicalsamples to the website; and (c) transmitting the corresponding pluralityof biological samples in the well plate to the screening laboratory. 37.The method of claim 28, wherein the step of showing includes the step ofshowing a corresponding row and column identifier for each of theplurality of wells.
 38. A method of screening genomic DNA in a pluralityof biological samples, wherein the samples are to be sent by a remoteuser for ultimate arrival at a screening laboratory, wherein thescreening is for a designated genetic sequence, and further wherein theremote user provides screening parameters via the Internet to ascreening website, the method comprising: (a) transmitting an accessenabling response from the screening website to the remote user via theInternet, the access enabling response including screening parameters;(b) receiving selected screening parameter selections via the Internetfrom the remote user; (c) receiving the plurality of biological samplesin a multi-well plate; (d) conducting screening of the plurality ofbiological samples according to the selected screening parameters withthe reagents to obtain screening results; and (e) transmitting screeningresults for the designated genetic sequence to the remote user via theInternet.
 39. The method of step 38 further comprising the step oftransmitting a request via the Internet to a supplier to obtain reagentsconforming to the screening parameter selections.
 40. The method ofclaim 38 wherein the screening parameter selections include a selectablemarker.
 41. The method of claim 38 wherein the plurality of biologicalsamples in a multi-well plate include at least two lines, and furtherwherein the screening parameter selections include identification of theat least two lines.
 42. The method of claim 38 wherein the screeningparameter selections include a probe sequence.
 43. The method of claim38 wherein the screening parameter selections include a designatedcontrol.
 44. The method of claim 43 wherein the designated control is abiological sample having the designated genetic sequence.
 45. The methodof claim 38 wherein the step of conducting screening includes the stepof treating the plurality of biological samples with a lysis buffer toobtain cellular debris including genomic DNA.
 46. The method of claim45, wherein the step of conducting screening further includes the stepof separating the genomic DNA from the cellular debris of the pluralityof biological samples using magnetic particles.
 47. The method of claim38 wherein the reagents are labeled probes specific for a portion of thedesignated genetic sequence.
 48. The method of claim 38 wherein therequest to a supplier is generated to an order manager.
 49. Acomputer-implemented method of processing an order for transgenic ortargeted mutagenesis screening of a plurality of biological samples in amulti-well plate by electronic communications between a website and aremote user's computer, the method comprising the steps of: (a)receiving from the remote user's computer a user account identifier andpassword; (b) checking validity of the account identifier and password;(c) receiving from the remote user a designation of a genetic sequencefor screening the plurality of biological samples; (d) receiving adesignation from the remote user's computer of a plurality of biologicalsamples for transgenic or targeted mutagenic screening; and (e)receiving from the remote user's computer a designation of which of theplurality of biological samples was placed into each well of amulti-well plate.
 50. The method of claim 49 wherein the designatedgenetic sequence is selected from the group including SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:
 5. 51. The methodof claim 49 wherein the designated genetic sequence is a transgenicinsert.
 52. The method of claim 49 wherein the designated geneticsequence is a selectable marker.
 53. The method of claim 49 wherein thedesignated genetic sequence is a knock-in.
 54. The method of claim 49wherein the designated genetic sequence is a knock-out.
 55. The methodof claim 49 further comprising the step of adding labeled probesspecific for reference genetic sequence to the plurality of biologicalsamples.
 56. The method of claim 49 wherein the reference geneticsequence is selected from the group consisting of: SEQ. ID NO: 6, SEQ.ID NO: 7, SEQ. ID NO: 8, SEQ. ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ IDNO:
 16. 57. The method of claim 49 further comprising the step ofreceiving selected screening parameter selections via the Internet fromthe remote user.
 58. The method of claim 57 wherein the screeningparameter selections include a selectable marker.
 59. The method ofclaim 57 wherein the screening parameter selections include adesignation of a plurality of lines to be tested.
 60. The method ofclaim 57 where the screening parameter selections includes a probesequence.
 61. The method of claim 57 wherein the screening parameterselections include a designated control.
 62. The method of claim 49further comprising the step of receiving a designation of a controlsample from the remote user via the Internet.
 63. The method of claim 62wherein the step of receiving a designation of a control sample includesthe step of receiving an identification of a zygosity of the controlsample from the remote user via the Internet.
 64. The method of claim 62wherein the step of receiving a designation of a control sample includesthe step of receiving an identification of a copy number of the controlsample from the remote user via the Internet.
 65. The method of claim 62wherein the step of receiving a designation of a control sample includesthe step of receiving an identification of a mosaic nature of thecontrol sample from the remote user via the Internet.
 66. The method ofclaim 49 further comprising the step of receiving a designation of aprobe sequence complimentary to a portion of the designated geneticsequence.
 67. The method of claim 49 further comprising the step oftransmitting screening results to the remote user, wherein the screeningresults are the result of screening the plurality of biological samples.68. The method of claim 67 wherein the step of transmitting screeningresults includes the step of transmitting a sample outcome report forthe plurality of biological samples to the remote user via the Internet.69. The method of claim 68 wherein the sample outcome report includes apictorial representation of screening results.
 70. The method of claim68 wherein the sample outcome report includes a quantitative analysis ofeach of the plurality of biological samples in comparison to adesignated control.
 71. The method of claim 68 wherein the sampleoutcome report includes a qualitative analysis of each of the pluralityof biological samples in comparison to a designated control.
 72. Themethod of claim 70, wherein the designated control includes thedesignated genetic sequence.
 73. The method of claim 68 wherein thesample outcome report includes an accession number of the well plate.74. The method of claim 68 wherein the sample outcome report includes awell location of each of the plurality of biological samples in the wellplate.
 75. The method of claim 68 wherein the sample outcome reportincludes a copy number for each of the plurality of biological samples.76. The method of claim 50 wherein the designated genetic sequence isselected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ IDNO: 3; SEQ ID NO: 4; and SEQ ID NO:
 5. 77. The method of claim 49further comprising the steps of: reviewing the designated geneticsequence; and designating a portion of the designated genetic sequenceas a target genetic sequence.
 78. The method of claim 77 furthercomprising the steps of: transmitting the target genetic sequence to asupplier to create a target-binding probe; and receiving thetarget-binding probe from the supplier.
 79. The method of claim 49wherein the plurality of biological samples comes from a genetic line.80. The method of claim 49, wherein the plurality of biological samplescomes from a plurality of genetic lines.
 81. A process for screening aplurality of biological samples in a multi-well plate at a screeninglaboratory, the process comprising the steps of: receiving an electroniccommunication from a remote user, the communication including screeningparameter selections; entering the screening parameter selections into alaboratory information management system of the screening laboratory;receiving the multi-well plate at the screening laboratory; andscreening genomic DNA isolated from the plurality of biological samplesat the screening laboratory.
 82. The process of claim 81 wherein thestep of receiving an electronic communication from a remote userincludes the step of receiving the electronic communication directlyfrom the remote user.
 83. The process of claim 81 wherein the step ofreceiving an electronic communication from a remote user includes thestep of receiving the electronic communication indirectly from theremote user.
 84. The process of claim 81 wherein the step of receivingelectronic communication from a remote user includes the step ofreceiving a designation of an electronic accession number of themulti-well plate, and further wherein the step of entering the screeningparameter selections includes the step of entering the designatedelectronic accession number into the laboratory information managementsystem.
 85. The process of claim 81 wherein the step of receiving themulti-well plate includes the step of electronically scanning an actualaccession number on the plate.
 86. The process of claim 85 wherein thestep of electronically scanning includes the step of barcode scanningthe actual accession number on the plate.
 87. The process of claim 86,wherein the step of barcode scanning the actual accession numberincludes the step of entering the barcode scanned accession number intothe laboratory information management system.
 88. The process of claim85 further comprising the step of comparing the actual accession numberwith the designated accession number previously entered by the remoteuser when the remote user ordered genetic scanning.
 89. The process ofclaim 88 further comprising the step of electronically communicating anarrival of the multi-well plate at the laboratory to the remote userafter the step of comparing.
 90. The process of claim 88 furthercomprising the steps of posting an arrival of the multi-well plate atthe laboratory on a web site after the step of comparing, wherein theremote user has password protected access to the web site.
 91. Theprocess of claim 88 further comprising the step of the e-mailing theremote user of an arrival of the plurality of biological samples in themulti-well plate at the laboratory after the step of comparingmulti-well plate accession numbers.
 92. The process of claim 81 whereinthe step of receiving an electronic communication from a remote userincludes the step of receiving the electronic communication from theremote user at the laboratory.
 93. The process of claim 81 wherein thescreening parameter selections include a selectable marker.
 94. Theprocess of claim 81 wherein the screening parameter selections includeidentifying the number of lines to be tested.
 95. The process of claim81 wherein the screening parameter selections include a designatedcontrol.
 96. The process of claim 81 wherein the screening parameterselections include a designated genetic sequence.
 97. The process ofclaim 96 further comprising the step of electronically transmitting thedesignated genetic sequence to a supplier.
 98. The process of claim 81further comprising the steps of: receiving an electronic designation ofa control sample from the remote user; and receiving the control sampleat the laboratory.
 99. The process of claim 98, wherein the step ofreceiving the control sample at the laboratory includes the step ofreceiving the control sample from the remote user.
 100. The process ofclaim 98 wherein the step of receiving an electronic designation of acontrol sample includes the step of receiving an electronicidentification of the zygosity of the control sample from the remoteuser via the Internet.
 101. The process of claim 98 wherein the step ofreceiving an electronic designation of a control sample includes thestep of receiving an electronic identification of a copy number of thecontrol sample from the remote user via the Internet.
 102. The processof claim 98 wherein the step of receiving an electronic designation of acontrol sample includes the step of receiving an electronicidentification of a mosaic nature of the control sample from the remoteuser via the Internet.
 103. The process of claim 98 further comprisingthe step of receiving an electronic designation of a probe sequencecomplimentary to a portion of the designated genetic sequence.
 104. Theprocess of claim 81 wherein each of the plurality of biological samplesis a tissue sample and further wherein each is contained in thecorresponding well of a plurality of wells of the multi-well plate, themethod further comprising the steps of: adding lysis buffer to each ofthe plurality of wells containing a tissue sample to lysis the tissue;and producing cellular debris including genomic DNA mixed with lysisbuffer in each of the plurality of wells.
 105. The process of claim 104further comprising the step of transferring the genomic DNA from each ofthe plurality of wells to a corresponding well in a second multi-wellplate.
 106. The process of claim 105 further comprising the step ofadding magnetic particles to each corresponding well in the secondmulti-well plate.